Substituted benzylidene indenyl formamides, acetamides and propionamides

ABSTRACT

Substituted benzylidene indenyl formamides, acetamides and propionamides are useful in the treatment of precancerous lesions and neoplasms.

This application is a continuation of U.S. patent application Ser. No.08/661,293, filed Jun. 13, 1996.

TECHNICAL FIELD

This invention relates to compounds and methods for treatment orprevention of precancerous lesions.

BACKGROUND OF THE INVENTION

Each year in the United States alone, untold numbers of people developprecancerous lesions. These lesions exhibit a strong tendency to developinto malignant tumors, or cancer. Such lesions include lesions of thebreast (that can develop into breast cancer), lesions of the skin (thatcan develop into malignant melanoma or basal cell carcinoma), colonicadenomatous polyps (that can develop into colon cancer), and other suchneoplasms. Compounds which prevent or induce the remission of existingprecancerous or cancerous lesions or carcinomas would greatly reduceillness and death from cancer.

For example, approximately 60,000 people die from colon cancer, and over150,000 new cases of colon cancer are diagnosed each year. For theAmerican population as a whole, individuals have a six percent lifetimerisk of developing colon cancer, making it the second most prevalentform of cancer in the country. Colon cancer is also prevalent in WesternEurope. It is believed that increased dietary fat consumption isincreasing the risk of colon cancer in Japan.

In addition, the incidence of colon cancer reportedly increases withage, particularly after the age of 40. Since the mean ages ofpopulations in America and Western Europe are increasing, the prevalenceof colorectal cancer should increase in the future.

To date, little progress has been made in the prevention and treatmentof colorectal cancer, as reflected by the lack of change in thefive-year survival rate over the last few decades. The only cure forthis cancer is surgery at an extremely early stage. Unfortunately, mostof these cancers are discovered too late for surgical cure. In manycases, the patient does not experience symptoms until the cancer hasprogressed to a malignant stage.

In view of these grim statistics, efforts in recent years haveconcentrated on colon cancer prevention. Colon cancer usually arisesfrom pre-existing benign neoplastic growths known as polyps. Preventionefforts have emphasized the identification and removal of colonicpolyps. Polyps are identified by x-ray and/or colonoscopy, and usuallyremoved by devices associated with the colonoscope. The increased use ofcolon x-rays and colonoscopies in recent years has detected clinicallysignificant precancerous polyps in four to six times the number ofindividuals per year that acquire colon cancer. During the past fiveyears alone, an estimated 3.5 to 5.5 million people in the United Stateshave been diagnosed with adenomatous colonic polyps, and it is estimatedthat many more people have or are susceptible to developing thiscondition, but are as yet undiagnosed. In fact, there are estimates that10-12 percent of people over the age of 40 will form clinicallysignificant adenomatous polyps.

Removal of polyps has been accomplished either with surgery orfiber-optic endoscopic polypectomy--procedures that are uncomfortable,costly (the cost of a single polypectomy ranges between $1,000 and$1,500 for endoscopic treatment and more for surgery), and involve asmall but significant risk of colon perforation. Overall, about $2.5billion is spent annually in the United States in colon cancer treatmentand prevention.

As indicated above, each polyp carries with it a chance that it willdevelop into a cancer. The likelihood of cancer is diminished if a polypis removed. However, many of these patients demonstrate a propensity fordeveloping additional polyps in the future. They must, therefore, bemonitored periodically for the rest of their lives for polypreoccurrence.

In most cases (i.e., the cases of so-called common sporadic polyps),polyp removal will be effective to reduce the risk of cancer. In a smallpercentage of cases (i.e., the cases of the so-called polyposissyndromes), removal of all or part of the colon is indicated. Thedifference between common sporadic polyps and polyposis syndromes isdramatic. Common sporadic polyp cases are characterized by relativelyfew polyps, each of which can usually be removed leaving the colonintact. By contrast, polyposis syndrome cases can be characterized bymany (e.g., hundreds or more) of polyps--literally covering the colon insome cases--making safe removal of the polyps impossible short ofsurgical removal of the colon.

Because each polyp carries with it the palpable risk of cancerousdevelopment, polyposis syndrome patients invariably develop cancer ifleft untreated. Surgical removal of the colon is the conventionaltreatment. Many of these patients have undergone a severe change inlifestyle as a result of the disfiguring surgery. Patients have strictdietary restrictions, and many must wear ostomy appliances to collecttheir intestinal wastes.

The search for drugs useful for treating and preventing cancer isintensive. Indeed, much of the focus of cancer research today is on theprevention of cancer because therapy is often not effective and hassevere side effects. Cancer prevention is important for recovered cancerpatients who retain a risk of cancer reoccurrence. Also, cancerprevention is important for people who have not yet had cancer, but havehereditary factors that place them at risk of developing cancer. Withthe development of new genetic screening technologies, it is easier toidentify those with high risk genetic factors, such as the potential forpolyposis syndrome, who would greatly benefit from chemopreventivedrugs. Therefore, finding such anti-cancer drugs that can be used forprolonged preventive use is of vital interest to many people.

One way to find such drugs is to screen thousands of compounds for thesame biological activity found in known chemopreventive andchemotherapeutic drugs.

Most such drugs are now believed to kill cancer cells by inducingapoptosis, or as sometimes referred to as "programmed cell death."Apoptosis naturally occurs in virtually all tissues of the body, andespecially in self-renewing tissues such as bone marrow, gut, and skin.Apoptosis plays a critical role in tissue homeostasis, that is, itensures that the number of new cells produced are correspondingly offsetby an equal number of cells that die. For example, the cells in theintestinal lining divide so rapidly that the body must eliminate cellsafter only three days in order to prevent the overgrowth of theintestinal lining.

Recently, scientists have realized that abnormalities of apoptosis canlead to the formation of precancerous lesions and carcinomas. Also,recent research indicates that defects in apoptosis play a major role inother diseases in addition to cancer. Consequently, compounds thatmodulate apoptosis could be used to prevent or control cancer, as wellas used in the treatment of other diseases.

Unfortunately, even though known chemotherapeutic drugs may exhibit suchdesirable apoptosis effects, most chemotherapeutic drugs have seriousside effects that prohibit their long term use, or use in otherwisehealthy individuals with precancerous lesions. These side effects, whichare a result of the high levels of cytotoxicity of the drugs, includehair loss, weight loss, vomiting and bone marrow immune suppression.Therefore, there is a need to identify new drug candidates for therapythat do not have such serious side effects in humans.

In the last few years, several non-steroidal anti-inflammatory drugs("NSAIDs"), originally developed to treat arthritis, have showneffectiveness in inhibiting and eliminating colonic polyps. Polypsvirtually disappear when the patients take the drug, particularly whenthe NSAID sulindac is administered. However, the prophylactic use ofcurrently available NSAIDs, even in polyposis syndrome patients, ismarked by severe side reactions that include gastrointestinalirritations and ulcerations, Once NSAID treatment is terminated due tosuch complications, the polyps return, particularly in polyposissyndrome patients.

Sulindac has been particularly well received among the NSAIDs for thepolyp treatment. Sulindac is a sulfoxide compound that itself isbelieved to be inactive as an anti-arthritic agent. The sulfoxide isreported to be converted by liver enzymes to the corresponding sulfide,which is acknowledged to be the active moiety as a prostaglandinsynthesis inhibitor. The sulfide, however, is associated with the sideeffects of conventional NSAIDs. The sulfoxide is also known to bemetabolized to sulfone compound that has been found to be inactive as aninhibitor of prostaglandin synthesis but active as an inhibitor ofprecancerous lesions.

SUMMARY OF THE INVENTION

This invention includes both pharmaceutical compositions containingcompounds and a method of treating patients with precancerous lesions byadministering a pharmacologically effective amount of those compoundsdescribed below to a patient in need of such treatment. Suchcompositions are effective in modulating apoptosis and eliminating andinhibiting the growth of precancerous lesions and neoplasms, but are notcharacterized by the severe side reactions of conventional NSAIDs.

The compounds used in the treatment of this invention are believed to beeffective on precancerous lesions either because they are activethemselves or because they are metabolized to active derivatives.

It was unexpectedly discovered that while the compounds of thisinvention do not greatly inhibit prostaglandin synthesis--prostaglandinsynthesis inhibition being a characteristic of conventional NSAIDs--thecompounds of this invention nonetheless have antiproliferative effectson precancerous lesion cells.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, the present invention provides a pharmaceuticalcomposition that includes compounds of formula I below for treating apatient with precancerous lesions: ##STR1## wherein R and R₁ areindependently selected from the group consisting of hydrogen, hydroxy,lower alkyl and amino; or R and R₁ together may be oxygen,

R₂, R₃ and R₄ are independently selected from the group consisting ofhydrogen, hydroxy, halogen, lower alkoxy, lower alkyl and alkylmercapto;

at least two of R₅, R₆ and R₇ are identically selected from the groupconsisting of hydroxy and lower alkoxy, and the third is selected fromthe group consisting of hydroxy, halogen, lower alkoxy, lower alkyl,amino and lower dialkylamino, with the proviso that when at least one ofR₂, R₃ or R₄ is lower alkoxy, then each of R₅, R₆ and R₇ are hydroxy orlower alkoxy;

R₈ is selected from the group consisting of hydrogen and lower alkyl;

o may be 0, 1 or 2; and

M is selected from the group consisting of amino, alkylamino,dialkylamino, alkoxyamino, alkenylamino, alkynylamino,hydroxyalkylamino, polyhydroxyalkylamino, dialkylaminoalkylamino,aminoalkylamino, arylalkylamino selected from the group consisting ofbenzylamino, anilino and phenylalkylamino, aminoindan, andheterocycloalkylamino where the heterocycles are selected from the groupconsisting of pyridinyl, piperidinyl, piperazinyl, pyrrollidinyl andN-morpholino; wherein the alkyl between the cyclic structure and theamino may be absent, and the cyclic structures may optionally besubstituted with one or more of halo, alkoxy, hydroxy, amino,alkylamino, dialkylamino and sulfonamido; or M may be NR'R" where NR' isas described above and R" is selected from alkyl, cyanoalkyl, haloalkyl,alkylamino, dialkylaminoalkyl, alkanoylalkylester and pyridinyl.

Preferably, o is 1 or 2; and M is selected from the group consisting ofamino, alkylamino, dialkylamino, alkenylamino, alkynylamino,hydroxyalkylamino, dialkylaminoalkylamino, arylalkylamino selected fromthe group consisting of benzylamino, anilino and phenylalkylamino,aminoindan, and heterocycloalkylamino where the heterocycles areselected from the group consisting of pyridinyl, piperidinyl,piperazinyl, and pyrrollidinyl; wherein the alkyl between the cyclicstructure and the amino may be absent, and the cyclic structures mayoptionally be substituted with one or more of halo, alkoxy, hydroxy,amino, alkylamino and dialkylamino; or M may be NR'R" where NR' is asdescribed above and R" is selected from alkyl, cyanoalkyl, alkylamino,dialkylaminoalkyl and alkanoylalkylester.

More preferably, M is selected from the group consisting ofalkenylamino, alkynylamino, benzylamino, and pyridinylalkylamino,wherein the cyclic structures may optionally be substituted with one ormore of halo, alkoxy, hydroxy, amino, alkylamino and dialkylamino; or Mmay be NR'R" where NR' is as described above and R" is selected fromalkyl, alkylamino and dialkylaminoalkyl.

Even more preferably, M is selected from the group consisting ofalkenylamino, alkynylamino, benzylamino, and pyridinylalkylamino,wherein the cyclic structures may optionally be substituted with one ormore of halo, alkoxy, hydroxy, amino, alkylamino and dialkylamino.

Still more preferably, M is selected from benzylamino and benzylaminosubstituted with one or more of halo, alkoxy, hydroxy, amino, alkylaminoand dialkylamino. Yet still more preferably, M is selected from thegroup consisting of benzylamino and benzylamino substituted with one ormore of halo, alkoxy and hydroxy. Most preferably, M is benzylamino.

For other substituents of the compound of formula I, when M is at leastin its even more preferred form, preferably, R and R₁ are independentlyselected from the group consisting of hydrogen and hydroxy; at least twoof R₅, R₆ and R₇ are lower alkoxy and the third is selected fromhydroxy, lower alkoxy, amino and lower dialkylamino; or each of R₅, R₆and R₇ are hydroxy.

More preferably, R and R₁ are hydrogen; R₂, R₃ and R₄ are independentlyselected from the group consisting of hydrogen, hydroxy, halogen, loweralkoxy, lower alkyl and alkyl mercapto; at least two of R₅, R₆ and R₇are lower alkoxy and the third is selected from hydroxy and loweralkoxy; R₈ is lower alkyl; and o is 1.

Even more preferably, R₅, R₆ and R₇ are each lower alkoxy; and R₈ ismethyl.

Still more preferably, R₂ is selected from the group consisting ofhydroxy, halogen, lower alkoxy and alkyl mercapto; R₄ is hydrogen; andR₅, R₆ and R₇ are each methoxy.

Yet still more preferably, R₂ is selected from the group consisting ofhydroxy, halogen and lower alkoxy; and R₃ is hydrogen. Even still morepreferably, R₂ is halogen, preferably fluoro.

A preferred group of compounds includes:

(Z)-5-Fluoro-2-methyl-1-(2,4,6-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide,

(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide;

(E)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide;

(Z)-5-Fluoro-2-methyl-1-(2,3,4-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide;and

(Z)-5-Fluoro-2-methyl-1-(2,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide.

Preferred compounds are(Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamideand the corresponding E-isomer. The most preferred compound is(Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide.

The present invention also is a method of treating a patient withprecancerous lesions by administering to a patient a pharmacologicallyeffective amount of a pharmaceutical composition that includes acompound of formula I, wherein R₁ through R₈ are as defined above.Preferably, this composition is administered without therapeutic amountsof an NSAID.

The present invention is also a method of treating individuals withprecancerous lesions by administering a pharmacologically effectiveamount of an enterically coated pharmaceutical composition that includescompounds of this invention.

Also, the present invention is a method of inhibiting the growth ofneoplastic cells by exposing the cells to an effective amount ofcompounds of formula I, wherein R₁ through R₈ are defined as above.

In still another form, the invention is a method of inducing apoptosisin human cells by exposing those cells to an effective amount ofcompounds of formula I, wherein R₁ through R₈ are defined as above wheresuch cells are sensitive to these compounds.

Additionally, in yet another form, the invention is a method of treatinga patient having a disease which would benefit from regulation ofapoptosis by treating the patient with an effective amount of compoundsof formula I, wherein R₁ through R₈ are defined as above. The regulationof apoptosis is believed to play an important role in diseasesassociated with abnormalities of cellular growth patterns such as benignprostatic hyperplasia, neurodegenerative diseases such as Parkinson'sdisease, autoimmune diseases including multiple sclerosis and rheumatoidarthritis, infectious diseases such as AIDS, and other diseases, aswell.

As used herein, the term "precancerous lesion" includes syndromesrepresented by abnormal neoplastic, including dysplastic, changes oftissue. Examples include adenomatous growths in colonic, breast, bladderor lung tissues, or conditions such as dysplastic nevus syndrome, aprecursor to malignant melanoma of the skin. Examples also include, inaddition to dysplastic nevus syndromes, polyposis syndromes, colonicpolyps, precancerous lesions of the cervix (i.e., cervical dysplasia),prostatic dysplasia, bronchial dysplasia, breast, bladder and/or skinand related conditions (e.g., actinic keratosis), whether the lesionsare clinically identifiable or not.

As used herein, the term "carcinomas" refers to lesions that arecancerous. Examples include malignant melanomas, breast cancer, prostatecancer and colon cancer.

As used herein, the term "neoplasm" refers to both precancerous andcancerous lesions.

As used herein, the term "halo" or "halogen" refers to chloro, bromo,fluoro and iodo groups, and the term "alkyl" refers to straight,branched or cyclic alkyl groups and to substituted aryl alkyl groups.The term "lower alkyl" refers to C₁ to C₈ alkyl groups.

Compounds of this invention may be formulated into compositions togetherwith pharmaceutically acceptable carriers for oral administration insolid or liquid form, or for rectal administration, although carriersfor oral administration are most preferred.

Pharmaceutically acceptable carriers for oral administration includecapsules, tablets, pills, powders, troches and granules. In such soliddosage forms, the carrier can comprise at least one inert diluent suchas sucrose, lactose or starch. Such carriers can also comprise, as isnormal practice, additional substances other than diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, troches and pills, the carriers may also comprise bufferingagents. Carriers such as tablets, pills and granules can be preparedwith enteric coatings on the surfaces of the tablets, pills or granules.Alternatively, the enterically coated compound can be pressed into atablet, pill, or granule, and the tablet, pill or granules foradministration to the patient. Preferred enteric coatings include thosethat dissolve or disintegrate at colonic pH such as shellac or EudragetS.

Pharmaceutically acceptable carriers include liquid dosage forms fororal administration, e.g., pharmaceutically acceptable emulsions,solutions, suspensions, syrups and elixirs containing inert diluentscommonly used in the art, such as water. Besides such inert diluents,compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, and sweetening, flavoring andperfuming agents.

Pharmaceutically acceptable carriers for rectal administration arepreferably suppositories which may contain, in addition to the compoundsof this invention excipients such as cocoa butter or a suppository wax.

The pharmaceutically acceptable carrier and compounds of this inventionare formulated into unit dosage forms for administration to a patient.The dosage levels of active ingredient (i.e., compounds of thisinvention) in the unit dosage may be varied so as to obtain an amount ofactive ingredient effective to achieve lesion-eliminating activity inaccordance with the desired method of administration (i.e., oral orrectal). The selected dosage level therefore depends upon the nature ofthe active compound administered, the route of administration, thedesired duration of treatment, and other factors. If desired, the unitdosage may be such that the daily requirement for active compound is inone dose, or divided among multiple doses for administration, e.g., twoto four times per day.

The pharmaceutical compositions of this invention are preferablypackaged in a container (e.g., a box or bottle, or both) with suitableprinted material (e.g., a package insert) containing indications,directions for use, etc.

The foregoing may be better understood from the following examples, thatare presented for purposes of illustration and are not intended to limitthe scope of the invention. As used in the following examples, thereferences to substituents such as R, R₁, R₂, etc., refer to thecorresponding compounds and substituents in the formula I above.

EXAMPLES EXAMPLE 1(Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxnbenzylidene)-3-(N-Benzyl)-indenylacetamide

(A) p-Fluoro-α-methylcinnamic acid.

p-Fluorobenzaldehyde (200 g, 1.61 mol), propionic anhydride (3.5 g, 2.42mol) and sodium propionate (155 g, 1.61 mol) are mixed in a one literthree-necked flask which had been flushed with nitrogen. The flask isheated gradually in an oil-bath to 140° C. After 20 hours, the flask iscooled to 100° C. and poured into 8 l of water. The precipitate isdissolved by adding potassium hydroxide (302 g) in 2 l of water. Theaqueous solution is extracted with ether, and the ether extracts washedwith potassium hydroxide solution. The combined aqueous layers arefiltered, are acidified with concentrated HCl, and are filtered. Thecollected solid, p-fluoro-(X-methylcinnamic acid, is washed with water,and is dried and used as obtained.

(B) p-Fluoro-α-methylhydrocinnamic acid.

To p-fluoro-α-methylcinnamic acid (177.9 g, 0.987 mol) in 3.6 l ethanolis added 11.0 g of 5% Pd/C. The mixture is reduced at room temperatureunder a hydrogen pressure of 40 p.s.i. When hydrogen uptake ceases, thecatalyst is filtered off, and the filtrate is concentrated in vacuo togive the product, p-fluoro-α-methylhydrocinnamic acid, which was usedwithout weighing in the next step.

(C) 6-Fluoro-2-methylindanone

To 932 g polyphosphoric acid at 70° C. (on the steam bath) is addedp-fluoro-α-methylhydrocinnamic acid (93.2 g, 0.5 mol) slowly withstirring. The temperature is gradually raised to 95° C., and the mixtureis kept at this temperature for 1 hour. The mixture is allowed to cooland added to 2l. of water. The aqueous layer is extracted with ether,the ether solution is washed twice with saturated sodium chloridesolution, 5% Na₂ CO₃ solution, water, and is then dried. The etherfiltrate is concentrated with 200 g silica-gel, and is added to a fivepound silica-gel column packed with 5% ether-petroleum ether. The columnis eluted with 5-10% ether-petroleum ether, to give6-fluoro-2-methylindanone. Elution is followed by TLC.

(D) 5-fluoro-2-methylindenyl-3-acetic acid

A mixture of 6-fluoro-2-methylindanone (18.4 g, 0.112 mol), cyanoaceticacid (10.5 g, 0.123 mol), acetic acid (6.6 g), and ammonium acetate (1.7g) in dry toluene (15.5 ml) is refluxed with stirring for 21 hours, asthe liberated water is collected in a Dean Stark trap. The toluene isconcentrated, and the residue dissolved in 60 ml of hot ethanol and 14ml of 2.2 N aqueous potassium hydroxide solution. 22 g of 85% KOH in 150ml of water is added, and the mixture refluxed for 13 hours undernitrogen. The ethanol is removed under vacuum, 500 ml water added, theaqueous solution is washed well with ether and then boiled withcharcoal. The aqueous filtrate is acidified to pH 2 with 50% coldhydrochloric acid. The precipitate and dried5-fluoro-2-methylindenyl-3-acetic acid (M.P. 164-166° C.) is obtained.

(E)(Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylaceticacid

5-fluoro-2-methyl-3-indenylacetic acid (15 g, 0.072 mol),3,4,5-trimethoxybenzaldehyde (17.85 g, 0.091 mol) and sodium methoxide(13.0 g, 0.24 mol) are heated in methanol (200 ml) at 60° C. undernitrogen with stirring for 6 hours. After cooling, the reaction mixtureis poured into 750 ml of ice-water, and is acidified with 2.5 Nhydrochloric acid. The collected solid is triturated with a little etherto produce(Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylaceticacid (M .P. 166-169° C.).

(F)(Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylacetylchloride

(Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylaceticacid(20.64 g, 54.70 mmol) in THF (150 ml) is allowed to react withoxalylchloride (2 M in CH₂ Cl₂ ; 35 ml; 70 mmol) under reflux conditions(24 h). The solvent is evaporated to yield the title compound, which isused as such in the next step.

(G)(Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide

Method I)

Benzylamine (0.55 ml, 5 mmol) is added slowly at room temperature to asolution of(Z)-5-fluoro-2-methyl-2-(3,4,5-trimethoxybenzylidene)-3-indenylacetylchloride (step F, 1 g, 2.5 mmol) in CH₂ Cl₂ (25 ml). The reactionmixture is refluxed overnight, and extracted with aqueous HCl (10%),water, and aqueous NaHCO₃ (5%). The organic phase is dried (Na₂ SO₄) andis evaporated to give the yellow title compound, which is recrystallizedfrom ethyl acetate to give the title compound (M.P. 191° C.).

(R=H, R₁ =H, R₂ =F, R₃ =H, R₄ =H, R₅ =OCH₃, R₆ =OCH₃, R₇ =OCH₃, R₈ =CH₃,o=1, M=PhCH₂ NH)

Method II)

(Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylaceticacid (1 g, 2.60 mmol) in DMA (5 ml) is allowed to react withN-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.77 g, 4mmol) and benzylamine (0.38 ml, 3.5 mmol) at room temperature for twodays. The reaction mixture is added dropwise to stirred ice-water (400ml). A yellow precipitate is filtered off, is washed with water (200ml), and is dried in vacuo. Recrystallization from ethyl acetate givesthe title compound.

Method III)

(Z)-5-fluoro-2-methylindene-3-(N-benzyl)-acetamide (1 g, 3.38 mmol),3,4,5-trimethoxybenzaldehyde (0.74 g, 4 mmol), and sodium methoxide (30ml, 1 M NaOCH₃ in methanol) are heated at 60° C. under nitrogen withstirring for 24 h. After cooling, the reaction mixture is poured intoice-water (200 ml). A yellow solid is filtered off, is washed with water(100 ml), and dried in vacuo. Recrystallization from ethyl acetate givesthe title compound.

EXAMPLES 2-51

When the methods for example 1(G) are followed but substituting forbenzylamine each of the amines listed in Table 1, there are obtained thecorresponding amides listed in Table 2.

                  TABLE 1                                                         ______________________________________                                        Example     Amine                                                             ______________________________________                                        2           3-iodobenzylamine                                                   3 3-chlorobenzylamine                                                         4 2-chlorobenzylamine                                                         5 3,4-dichlorobenzylamine                                                     6 2,4-difluorobenzylamine                                                     7 3-fluorobenzylamine                                                         8 2,5-difluorobenzylamine                                                     9 3,4-difluorobenzylamine                                                     10 2,6-difluorobenzylamine                                                    11 3-fluoro-5-(trifluoromethyl)-benzylamine                                   12 4-(trifluoromethyl)-benzylamine                                            13 4-(trifluoromethoxy)-benzylamine                                           14 4-methoxybenzylamine                                                       15 2-methoxybenzylamine                                                       16 3,5-dimethoxybenzylamine                                                   17 3,4-dimethoxybenzylamine                                                   18 2,3-dimethoxybenzylamine                                                   19 2,4-dimethoxybenzylamine                                                   20 4-(aminomethyl)-benzenesulfonamide                                         21 4-(dimethylamino)-benzylamine                                              22 aniline                                                                    23 4-bromoaniline                                                             24 diethylamine                                                               25 tert. butylamine                                                           26 dicyclohexylamine                                                          27 propargylamine                                                             28 allylamine                                                                 29 furfurylamine                                                              30 cyclopropylmethylamine                                                     31 N-benzylglycine ethyl ester                                                32 2-fluorobenzylamine                                                        33 2-bromobenzylamine                                                         34 2-aminobenzylamine                                                         35 2-(4-methoxybenzylamino)-pyridine                                          36 N-benzyl-N',N'-dimethylethylene diamine                                    37 3-(benzylamino)-propionitrile                                              38 2-benzylamino pyridine                                                     39 phenethylamine                                                             40 3-phenyl propylamine                                                       41 4-phenyl butylamine                                                        42 (R)-1-phenylethylamine                                                     43 (S)-1-phenylethylamine                                                     44 3-dimethylamino propylamine                                                45 (R)-2-amino-2-phenylethanol                                                46 (S)-2-amino-2-phenylethanol                                                47 N-benzyl ethanolamine                                                      48 2-amino methyl pyridine                                                    49 3-amino methyl pyridine                                                    50 4-amino methyl pyridine                                                    51 penta fluorobenzylamine                                                  ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Ex-                                                                             am-                                                                           ple Amide                                                                   ______________________________________                                        2    (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-3-                   iodobenzyl)-indenylacetamide                                             3 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-3-                    chlorobenzyl)-indenylacetamide                                           4 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-2-                    chlorobenzyl)-indenylacetamide                                           5 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-3,4-                  dichlorobenzyl)-indenylacetamide                                         6 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-2,4-                  difluorobenzyl)-indenylacetamide                                         7 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-3 -                   fluorobenzyl)-indenylacetamide                                           8 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3(N-2,5-                   difluorobenzyl)-indenylacetamide                                         9 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-3,4-                  difluorobenzyl)-indenylacetamide                                         10 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-2,6-                 difluorobenzyl)-indenylacetamide                                         11 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-3-                   fluoro-5-(trifluoromethyl)-benzyl)-indenylacetamide                      12 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-4-                   (trifluoromethyl)-benzyl)-indenylacetamide                               13 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-4-                   (trifluoromethoxy)-benzyl)-indenylacetamide                              14 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-4-                   methoxybenzyl)-indenylacetamide                                          15 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-2-                   methoxybenzyl)-indenylacetamide                                          16 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-3,5-                 dimethoxybenzyl)-indenylacetamide                                        17 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-3,4-                 dimethoxybenzyl)-indenylacetamide                                        18 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-2,3-                 dimethoxybenzyl)-indenylacetamide                                        19 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-2,4-                 dimethoxybenzyl)-indenylacetamide                                        20 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-4-                   sulfonamidobenzyl)-indenylacetamide                                      21 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-4-                   (dimethylamino)-benzyl)-indenylacetamide                                 22 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-                 phenyl)-indenylacetamide                                                     23 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-4-                   bromophenyl)-indenylacetamide                                            24 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N,N-                   diethyl)-indenylacetamide                                                25 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-                 tertbutyl)-indenylacetamide                                                  26 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N,N-                   dicyclohexyl)-indenylacetamide                                           27 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-                 propargyl)-indenylacetamide (M.P. 171-172° C.)                        28 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-allyl)-              indenylacetamide (M.P. 147-149° C.)                               29 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-                 furfuryl)-indenylacetamide (M.P. 166-167° C.)                         30 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-                 cyclopropylmethyl)-indenylacetamide (M.P. 150-151° C.)                31 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-                 benzyl-N-glycinylethyl ester)-indenylacetamide                               32 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-2-                   fluorobenzyl)-indenylacetamide                                           33 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-2-                   bromobenzyl)-indenylacetamide                                            34 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-2-                   aminobenzyl)-indenylacetamide                                            35 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-4-                   methoxybenzyl-N-(2-pyridinyl))-indenylacetamide                          36 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-                 benzyl-N-2-dimethylaminoethyl)-indenylacetamide                              37 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-                 benzyl-N-(2-cyanoethyl))-indenylacetamide                                    38 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-                 benzyl-N-(2-pyridinyl))-indenylacetamide                                     39 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-2-                   phenylethyl)-indenylacetamide                                            40 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-3-                   phenylpropyl)-indenylacetamide                                           41 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-4-                   phenylbutyl)-indenylacetamide                                            42 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-(R)-1-               phenylethyl)-indenylacetamide                                            43 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-(S)-1-               phenylethyl)-indenylacetamide                                            44 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-3-                   dimethylaminopropyl)-idenylacetamide                                     45 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-(R)-1-               hydroxy-2-phenylethyl)-idenylacetamide                                   46 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-(S)-1-               hydroxy-2-phenylethyl)-idenylacetamide                                   47 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-                 benzyl-N-(1-hydroxyethyl))-idenylacetamide                                   48 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-2-                   pyridinyl)-idenylacetamide                                               49 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-3-                   pyridinyl)-idenylacetamide                                               50 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-4-                   pyridinyl)-idenylacetamide                                               51 (Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N -                    pentafluorobenzyl)-idenylacetamide (MP 209-210° C.)             ______________________________________                                    

EXAMPLE 52

(E)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide

When the residue from the evaporated mother liquor of preparation 1(E)is recrystallized from CH₃ CN, there is obtained(E)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylaceticacid, which is allowed to react with benzylamine according to theprocedures of example 1 to give(E)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-idenylacetamide,M.P. 149-152° C.

EXAMPLES 53-63

According to the procedures in Example 1, substituted benzaldehydes maybe used in place of 3,4,5-trimethoxybenzaldehyde in step (E) at the samereaction conditions to obtain the corresponding acids. These acids maybe used in place of(Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylaceticacid in Example 1, steps (F) and (G), to obtain the correspondingbenzylamides as listed in the examples below.

53) 2,4-dimethoxybenzaldehyde

(Z)-5-fluoro-2-methyl-1-(2,4-dimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide(R and R₁ =H, R₂ =F, R₃ and R₄ =H, R₅ and R₆ =OCH₃, R₇ =H, R₈ =CH₃, o=1,M=PhCH₂ NH)

54) 3,5-dimethoxybenzaldehyde

(Z)-5-fluoro-2-methyl-1-(3,5-dimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide(R and R₁ =H, R₂ =F, R₃ and R₄ =H, R₅ =H, R₆ and R₇ =OCH₃, R₈ =CH₃, o=1,M=PhCH₂ NH)

55) 2,4,6-trimethoxybenzaldehyde

(Z)-5-fluoro-2-methyl-1-(2,4,6-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide (R and R₁ =H, R₂ =F, R₃ and R₄ =H, R₅, R₆ an R₇=OCH₃, R₈ =CH₃, o=1, M=PhCH₂ NH) M.P. 224-225° C.

56) 4-chloro-3,5-dimethoxybenzaldehyde

(Z)-5-fluoro-2-methyl-1-(4-chloro-3,5-dimethoxy-benzylidene)-3-(N-benzyl)-indenylacetamide(R and R₁ =H, R₂ =F, R₃ and R₄ =H, R₅ =OCH₃, R₆ =Cl, R₇ =OCH₃, R₈ =CH₃,o=1, M=PhCH₂ NH)

57) 2,3,4-trimethoxybenzaldehyde

(Z)-5-fluoro-2-methyl-1-(2,3,4-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide(R and R₁ =H, R₂ =F, R₃ and R₄ =H, R₅, R₆ and R₇ =OCH₃, R₈ =CH₃, o=1,M=PhCH₂ NH) M.P. 147-148° C.

58) 2,4,5-trimethoxybenzaldehyde

(Z)-5-fluoro-2-methyl-1-(2,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide(R and R₁ =H, R₂ =F, R₃ and R₄ =H, R₅, R₆ and R₇ =OCH₃, R₈ =CH₃, o=1,M=PhCH₂ NH) M.P. 203-204° C.

59) 4-methyl-2,3-dimethoxybenzaldehyde

(Z)-5-fluoro-2-methyl-1-(4-methyl-2,3-dimethoxy-benzylidene)-3-(N-benzyl)-indenylacetamide(R and R₁ =H, R₂ =F, R₃ and R₄ =H, R₅ and R₆ =OCH₃, R₇ =CH₃, R₈ =CH₃,o=1, M=PhCH₂ NH)

EXAMPLE 605-Methoxy-2-Methyl-1-(2,3.4-Trimethoxybenzylidene)-3-(N-benzal)-indenylacetamide

(A) α-Methyl-β-(p-methylthiophenyl) propionic acid

To a solution of 2.3 g (0.1 mol) of sodium in 100 ml of absolute alcoholis added 17.4 g (0.1 mol) of diethyl methylmalonate and 17.3 g (0.1 mol)of p-methylthiobenzylchloride. The mixture is heated under a reflux in awater bath for three hours. The reaction mixture is poured into water,and the aqueous solution is extracted six times with ether and dried. Itis then evaporated to yield diethyl methyl-p-methylthiobenzyl malonate.The crude product is then saponified by heating with excess 4% sodiumhydroxide in aqueous ethanolic solution. The solution thus formed isconcentrated, extracted with ether to remove any neutral material, andacidified with dilute sulfuric acid. The acidic mixture is heated on asteam bath for one hour, cooled and then extracted with ether.Evaporation of the ether solution gives α-methyl-β-(p-methylthiophenyl)propionic acid.

With other substituted malonic esters in place of diethyl methylmalonateand other substituted benzyl halides in place of p-methylthiobenzoylchloride, the corresponding substituted propionic acids are obtained, ina similar manner, for example:

α-methyl-β-(p-methoxyphenyl)propionic acid,

α-allyl-β-(p-nitrophenyl)propionic acid.

(B) 6-methoxy-2-methylindanone

α-Methyl-β-(p-methoxyphenyl)propionic acid (15 g) is added topolyphosphoric acid (170 g) at 50° C. and the mixture is heated at83-90° C. for two hours. The syrup is poured into iced water, stirredfor one-half hour, and then extracted with ether three times. The ethersolution is washed with water twice, and with 5% NaHCO₃ five times untilall the acidic material has been removed. The remaining neutral solutionis washed with water and dried over sodium sulfate. Evaporation of thesolution gives the indanone as a pale yellow oil.

Other β-aryl propionic acid compounds are converted to the correspondingindanone by the procedure of this example in a similar manner.

(C) Methyl 5-methoxy-2-methyl-3-indenylacetate.

A solution of 13.4 g of 6-methoxy-2-methylindanone and 19.3 g of methylbromoacetate in 45 ml benzene is added over a period of 5 minutes to 21g of zinc amalgam (prepared according to Organic Synthesis, Coll. Vol.3, p. 444) in 110 ml benzene and 40 ml dry ether. A few crystals ofiodine are added to start the reaction, and the reaction mixture ismaintained at reflux temperature (ca. 65° C.). At 3 hour intervals, twobatches of 10 g zinc amalgam and 10 g methyl bromo acetate are added.The mixture is then refluxed for 8 hours. After addition of 30 mlethanol and 150 ml of acetic acid, the mixture is poured into 700 ml of50% aqueous acetic acid. The organic layer is separated, and the aqueouslayer is extracted twice with ether. The combined organic layers arewashed thoroughly with water, ammonium hydroxide and water.

Drying over sodium sulfate, evaporation of solvent in vacuo, followed bypumping at 80° C. (bath temp.) (1-2 mm.) gives crudemethyl(1-hydroxy-2-methyl-methoxy-indenyl)acetate.

A mixture of the above crude hydroxyester, 20 g of p-toluenesulfonicacid monohydrate and 20 g of anhydrous calcium chloride in 250 mltoluene is refluxed overnight. The solution is filtered, and the solidresidue is washed with benzene. The combined benzene solution is washedwith water, sodium bicarbonate, and water, and is then dried over sodiumsulfate. After evaporation, the residual crude methyl5-methoxy-2-methyl-3-indenylacetate is chromatographed on acid-washedalumina, and the product is eluted with petroleum ether-ether (v./v.50-100%).

METHYL 2,6-DIMETHYL-3-INDENYLACETATE

The above reactions of Example 60(C) are repeated except that thestarting materials are 2,5-dimethylindanone and methylbromoacetate.Using the same reaction conditions and techniques there is obtainedmethyl 2,6-dimethyl-3-indenylacetate.

The above reaction s of Example 60(C) are repeated except that thestarting materials are 6-methylthioindanone and methybromoacetate. Usingthe same reaction conditions and techniques, there is obtained methyl5-methyl-thio-2-methyl-3-indenyl-acetate.

When any of the other indanones described in the other examples of thespecification are used in the above procedure in place of6-methoxy-2-methylindanone the corresponding methyl ester is obtained.

(D) 5-methoxy-2-methyl-1-(2,3,4-trimethoxybenzylidene)-3-indenyl aceticacid.

To a solution of methyl 5-methoxy-2-methyl-3-indenylacetate 8.7 g (0.037mol) and 2,3,4-trimethoxybenzaldehyde, 7.99 g (1.1 equivalent) is added16+ ml (2.0+ equivalents) of 25% methanolic sodium methoxide. Themixture is stirred at reflux under nitrogen for 2 hours. An equal volumeof water is added dropwise and refluxing continues for 30 min. Thesolution is cooled, diluted with water and extracted with ether (3×).Residual ether is blown off with nitrogen, and then the aqueous solutionis acidified with 50% glacial acetic acid. The precipitated product iscollected and washed thoroughly with water. The crude product iscrystallized to give5-methoxy-2-methyl-1-(2,3,4-trimethoxybenzylidene)-3-indenyl aceticacid.

(E)5-methoxy-2-methyl-1-(2,3,4-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide

Following the procedures of Example 1(F) and (G) and using the aciddescribed above leads to the formation of the title compound. (R and R₁=H, R₂ =OCH₃, R₃ and R₄ =H, R₅, R₆ and R₇ =OCH₃, R₈ =CH₃, o=1, M=PhCH₂NH)

EXAMPLE 615-Methoxy-2-Methyl-1-(3,4,5-Trimethoxy-Benzylidene)-3-(N-Benzyl)-Indenylacetamide

The above reaction of Example 60(D) and (E) is repeated with3,4,5-trimethoxybenzaldehyde instead of 2,3,4-trimethoxy-benzaldehyde.With the same reaction conditions and techniques,5-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamideis obtained. (R and R₁ =H, R₂ =OCH₃, R₃ and R₄ =H, R₅, R₆ and R₇ =OCH₃,R₈ =CH₃, o=1, M=PhCH₂ NH)

EXAMPLE 625-Hydroxy-2-Methyl-1-(2,4,6-Trimethoxy-Benzylidene)-3-(N-Benzyl)-Indenylacetamide

The reactions of Example 60(D) and (E) are repeated with methyl5-hydroxy-2-methyl-3-indenylacetate and 2,4,6-trimethoxybenzaldehyde asstarting, materials, and5-hydroxy-2-methyl-1-(2,4,6-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamideis obtained. (R and R₁ =H, R₂ ==OH, R₃ and R₄ =H, R₅, R₆ and R₇ =OCH₃,R₈ =CH₃, o=1, M=PhCH₂ NH)

The other methyl esters of Example 60(C) react with2,4,6-trimethoxybenzaldehyde according to the above procedure to producethe corresponding (N-benzyl) indenyl acetamide.

EXAMPLE 635-Methoxy-2-Methyl-1-(2,4,5-Trimethoxybenzylidene)-3-(N-Benzyl)-IndenylAcetamide

(A) 6-methoxy-2-methylindanone.

In a 500 ml 3-necked flask is placed 36.2 g (0.55 mol) of zinc dust, anda 250 ml addition funnel is charged with a mixture of 80 ml anhydrousbenzene, 20 ml of anhydrous ether, 80 g (0.58 mol) of p-anisaldehyde and98 g (0.55 mol) of ethyl-2-bromopropionate. About 10 ml of the mixtureis added to the zinc dust with vigorous stirring, and the suspension iswarmed gently until an exothermic reaction commences. The remainingreactants are added dropwise at such a rate that the reaction mixture isrefluxing smoothly on its own accord (ca. 30-35 min.). After addition iscompleted, the flask is placed in a water bath, the suspension is heatedat reflux for 30 minutes, and is cooled to 0° C. Sulfuric acid (250 ml,10%) is added with vigorous stirring. The benzene layer is extractedtwice with 50 ml portions of 5% sulfuric acid and washed with 50 mlportions of sulfuric acid (2×) and 50 ml portions of water (2×). Theaqueous acidic layers are combined and extracted with 2×50 ml ether. Thecombined ether and benzene extracts are dried over sodium sulfate.Evaporation of solvent and fractionation of the residue through a 6"Vigreux column affords the product,ethyl-2-hydroxy-(p-methoxyphenyl)-1-methylpropionate, B.P. 155-160° C.(1.5 mm).

By the method described in Vanden Zanden, Rec. trav. chim., 68, 413(1949), the above compound is converted to 6-methoxy-2-methylindanone.

5-ETHYL-2-METHYLINDANONE

The above reactions of Example 63(A) are repeated except that thestarting materials are o-ethylbenzaldehyde and ethyl-2-bromopropionate.By using the same reaction conditions and techniques, there is obtained5-ethyl-2-methylindanone.

When the benzaldehydes listed in Table 3 below are used in the procedureof Example 63A, the corresponding indanone is obtained.

                  TABLE 3                                                         ______________________________________                                        Aldehyde         Indanone                                                     ______________________________________                                        p,-o-, or m-tolualdehyde                                                                       2,6-dimethyl,                                                   2,5-dimethyl, or                                                              2,4-dimethyl-indanone                                                        p,-o-, or m-hydroxybenzaldehyde 4, 5 or 6-hydroxy-2-methylindanone                            p,-o-, or nitrobenzaldhyde 2-methyl-(4, 5 or 6)                              nitroindanone                                                  p,-o-, or m-chlorobenzaldehyde (4, 5, or 6)-chloro-2-methylindanone                           p,-o-, or m-cyanobenzaldehyde (4, 5, or 6)-cyano-2-methy                     lindanone                                                      vanillin 6-hydroxy-5-methoxy-                                                  2-methylindanone                                                             p,-o-, or m-sulfamylbenzaldehyde 2-methyl-(4,5 or 6)-sulfamylindanone                         3-chloro-4-methylbenzaldehyde 5-chloro-2,6 dimethylindan                     one                                                            4-carbamide-5-methylbenaldehyde 6-carbomide-2,5 dimethylindanone                              3,4-difluorobenzaldehyde 5,6-difluoro-2-methylindanone                        3,4,5-trifluorobenzaldehyde 5,6,7-trifluoro-2-methylinda                     none                                                         ______________________________________                                    

(B)5-methoxy-2-methyl-1-(2,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide

The reactions of Examples 60(C)-(E) are repeated, and5-methoxy-2-methyl-1-(2,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamideis obtained.

EXAMPLE 641-(3,4,5-Trimethoxybenzylidene)-2-Methyl-5-Methoxy-3-Indenyl-(N-Benzyl)-Propionamide

(A) Methyl-α(5-methoxy-2-methyl-3-indenyl) propionate.

The procedure of Example 60(C) is followed using methylα-bromopropionate in equivalent quantities in place of methylbromoacetate used therein. There is obtained methylα-(1-hydroxy-6-methoxy-2-methyl-1-indenyl)propionate, and it is thendehydrated to methyl α-(5-methoxy-2-methyl-3-indenyl)propionate in thesame manner.

(B) α-[1-(3,4,5-trimethoxybenzylidene)-2-methyl-5-methoxy-3-indenyl]propionic acid

To a solution of 0.5 g (1.92 mmol) of methylα-(5-methoxy-2-methyl-3-indenyl) propionate and 0.77 g (3.9 mmol) of3,4,5-trimethoxybenzaldehyde in 3 ml of anhydrous pyridine is added 1.63g of a 40% solution of benzyltrimethylammonium hydroxide (Triton-B) inmethanol. The resulting red-purple solution is stirred at roomtemperature overnight.

The reaction mixture is poured into a mixture of ice and water,acidified with 2.5 N HCl, and extracted with ether. The ether solutionis then washed with 2.5 N HCl until the washing acidifies (once), thenwith water until neutral. The ether layer is then extracted with 5% Na₂CO₃ solution. The Na₂ CO₃ solution is washed with ether, acidified andextracted with ether. The ether solution is washed with water, driedover Na₂ SO₄ and concentrated in vacuo to obtainα-[1-(3,4,5-trimethoxybenzylidene)-2-methyl-5-methoxy-3-indenyl]propionic acid.

(C) 1-(3,4,5-Trimethoxybenzylidene)-2-methyl-5-methoxy-3-indenyl-(N-benzyl)-propionamide

Following the procedure of Example 1(G) and using the acid describedabove leads to the formation of the title compound. (R=CH₃, R₁ =H, R₂=OCH₃, R₃ and R₄ =H, R₅, R₆ and R₇ =OCH₃, R₈ =CH₃, o=1, M=PhCH₂ NH)

EXAMPLE 651-(2,4,6-Trimethoxybenzylidene)-5-Dimethylamino-2-Methyl-3-(N-Benzyl)-IndenylAcetamide

(A) Methyl-3-hydroxy-2-methyl-5-nitro-3-indenylacetate

The procedure of Example 60(C) is followed using 2-methyl-6-nitroindanone in equivalent quantities in place of6-methyoxy-2-methyl-indanone used therein. After the mixture iscondensed, 30 ml of ethanol and 50 ml of acetic acid are added. Themixture is then poured into 700 ml of water. Extraction with ether givesmethyl 3-hydroxy-2-methyl-5-nitro-3-indenylacetate.

(B) Methyl 5-dimethylamino-2-methyl-3-indenylacetate.

A solution of 0.05 mol of methyl3-hydroxy-2-methyl-5-nitro-3-indenylacetate, 0.2 mol of 38% aqueousformaldehyde and 2 ml of acetic acid in 100 ml ethanol is reducedcatalytically in the presence of a 10% Pd/C catalyst under 40 lb p.s.i.hydrogen pressure at room temperature. The solution is filtered,evaporated and chromatographed on 300 g of silica gel to give methyl5-dimethylamino-3-hydroxy-2-methyl-3-indenylacetate. The hydroxy esteris then dehydrated to methyl 5-dimethylamino-2-methyl-3-indenylacetate.

(C) 1-(2,4,6-trimethoxybenzylidene)-5-dimethylamino-2-methyl -3-indenylacetic acid.

To a solution of 2.5 g of the ester from Part B of this example in 15 mlof 1,2-dimethoxyethane at 0° C. is added 1.95 g of2,4,6-trimethoxybenzaldehyde followed by 1.1 g of potassium t-butoxide.The reaction mixture is kept in the ice-bath for 4 hours, and thenallowed to stand at room temperature for 18 hours. The mixture isdiluted with 15 ml of ether and the potassium salt is filtered. The saltis dissolved in 30 ml of water and neutralized with dilute hydrochloricacid to pH 6-6.5. The crude acid precipitated is collected by filtrationand re-crystallized to give1-(2,4,6-trimethoxybenzylidene)-5-dimethylamino-2-methyl-3-indenylacetic acid.

(D)1-(2,4,6-Trimethoxybenzylidene)-5-dimethylamino-2-methyl-3-(N-benzyl)-indenylacetamide

Following the procedure of Example 1(G) and using the acid describedabove leads to the formation of the title compound. (R and R₁ =H, R₂=N(CH₃)₂, R₃ and R₄ =H, R₅, R₆ and R₇ =OCH₃, R₈ =CH₃, o=1, M=PhCH₂ NH)

EXAMPLE 66α-[1-(3,4,5-Trimethoxybenzylidene)-2-Methyl-5-Dimethylamino-3-Indenyl]-(N-Benzyl)-Propionamide

(A)α-[1-(3,4,5-trimethoxybenzylidene)-2-methyl-5-dimethylamino-3-indenyl]-propionicacid.

The procedures of Examples 63(A) and (B) are followed using6-dimethylamino-2-methylindanone in place of 6-methoxy-2-methylindanoneand methyl-α-bromopropionate in place of methyl bromoacetate usedtherein. There is obtainedα-[1-(3,4,5-trimethoxybenzylidene)-2-methyl-5-dimethylamino-3-indenyl]-(N-benzyl)-propionamide.(R=CH₃, R₁ =H, R₂ =N(CH₃)₂, R₃ and R₄ =H, R₅, R₆ and R₇ =OCH₃, R₈ =CH₃,o=1, M=PhCH₂ NH)

EXAMPLE 675,6-Difluoro-2-Methyl-1-(3,4,5-Trimethoxybenzylidene)-3-(N-Benzyl)-IndenylAcetamide

(A) 3,4-difluorobenzaldehyde,

In a 250 ml three-necked flask equipped with a magnetic stirrer,thermometer, condenser, and dropping funnel is placed 25.6 g (0.2 mol)of 3,4 difluorotoluene. The liquid is heated to 105° C. and illuminatedas 67 g (0.42 mol) of bromine is added slowly. The temperature is keptbetween 105-110° C. while the first half of the bromine is added over aperiod of one hour. The rest of the bromine is added over approximatelya 2-hour period and the temperature is raised to 150° C. and kept therefor 5 minutes.

The reaction mixture is cooled and transferred to a 1 liter 3-neckedflask with a motor driven stirrer and condenser. 120 ml H₂ O and 90 g ofcalcium carbonate are added, and the mixture is refluxed for 20 hourswith good stirring. The reaction mixture is steam distilled until nofurther oil is collected. The oil is taken up in methylene chloride anddried over MgSO₄. Evaporation of the solvent yields3,4-difluorobenzaldehyde which is used without further purification.

(B) 3,4-difluoro-α-methylcinnamic acid.

A mixture of 2.88 g (0.02 mol) of 3,4-difluorobenzaldehyde, 3.24 g(0.025 mol) of propionic anhydride and 0.92 g (0.02 mol) of sodiumpropionate under nitrogen is heated at 135° C. with a magnetic stirrerfor 20 hours. The reaction mixture is poured onto 50 ml of water. Asolid precipitates that dissolves when 50 ml of saturated K₂ CO₃ isadded with stirring. The basic solution is extracted with ether (2×100ml). The aqueous phase is then poured into an excess of concentrated HCland ice. The precipitated white solid is filtered and dried to give3,4-difluoro-ox-methylcinnamic acid, M.P. 122-125° C.

4-TRIFLUOROMETHYL-α-METHYLCINNAMIC ACID

The above reaction of Example 67(B) is repeated except that4-trifluoromethylbenzaldehyde is used as a starting material in place of3,4-difluorobenzaldehyde. Using the same reaction conditions andtechniques there is obtained 4-trifluoromethyl-α-methylcinnamic acid.

By similarly using other benzaldehydes such as 4-methylthiobenzaldehyde,4-chlorobenzaldehyde, and 3-methyl-4-chlorobenzaldehyde, there isobtained 4-methylthio-α-methylcinnamic acid, 4-chloro-α-methylcinnamicacid and 3-methyl-4-chloro-α-methylcinnamic acid respectively.

(C) 3,4-difluoro-α-methylhydrocinnamic acid.

A mixture of 28 g (0.141 mol) of 3,4-difluoro-ot-methylcinnamic acid, 1g of PtO₂, and 250 ml of MeOH is hydrogenated at 45 p.s.i. until thetheoretical uptake is completed. The catalyst is filtered off, and thematerial evaporated to one-third its volume. A 15% potassium hydroxidesolution (10 ml) is added, and the mixture refluxed for 30 minutes, ispoured into water, and is extracted with ether (2×100 ml). The aqueouslayer is acidified with concentrated HCl and ice. The oil whichseparates is extracted into ether, the ether solution dried over MgSO₄and evaporated to leave a clear oil which crystallizes.3,4-difluoro-α-methylhydrocinnamic acid, M.P. 55-56° C., is isolated.

(D) 5,6-difluoro-2-methyl-1-indanone.

20 g (0.1 mol) of 3,4-difluoro-α-methylhydrocinnamic acid is added to250 g of polyphosphoric acid. The mixture is efficiently stirred andheated on a steam bath for 2 hours. The mixture is poured onto ice-water(400 ml). The precipitate is extracted with ether (3×100 ml). Theextract is washed with saturated potassium carbonate, water and thendried (MgSO₄). The ether solution, when evaporated, leaves solid5,6-difluoro-2-methyl-1-indanone (M.P. 66-68° C.) which is used withoutfurther purification.

(E) 5,6-difluoro-2-methylindene-3-acetic acid methyl ester.

A mixture of 9.1 g (0.05 mol) of 5,6-difluoro-2-methyl-1-indanone, 4.0 gof "activated" zinc dust, 7.6 g (0.05 mol) of methyl bromoacetate, and acrystal of iodine in 250 ml of dry benzene is refluxed for 4-5 hours.TLC (20% Et₂ O-80% pet. ether on Si gel) shows greater than 95%conversion at this time. The reaction mixture is poured onto 250 ml of5% H₂ SO₄ The organic phase was dried (MgSO₄). Removal of solvent leavesan oily hydroxy ester, which is redissolved in 100 ml of benzene, and isrefluxed with phosphorus pentoxide (20 g) for 30 minutes. The organiclayer is decanted, the residue is washed with benzene, and the combinedorganic layers are washed with water (2×100 ml) and dried (MgSO₄). Thebenzene, when evaporated, leaves 5,6-difluoro-2-methylindene-.3-aceticacid methyl ester, M.P. 86-90° C.

5-METHYLTHIO-2-METHYLINDENE-3-ACETIC ACID METHYL ESTER

The above reaction of Example 67(E) is repeated using5-methylthio-2-methylindanone instead of5,6-difluoro-2-methyl-l-indanone. Using the same conditions andtechniques, there is obtained 5-methylthio-2-methylindene-3-acetic acidmethyl ester.

When an acylamino or sulfonyl indanone is employed as the startingmaterial in the above procedure, the corresponding methyl ester isobtained.

(F)5,6-difluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-indenyl-3-aceticacid.

1.19 g (5.0 mmol) of 5,6-difluoro-2-methylindene-3-acetic acid methylester is dissolved in 10 ml of dry pyridine followed by 0.98 g (5.0mmol) of 3,4,5-trimethoxybenzaldehyde. The flask is placed undernitrogen, and 5.0 g (5.1 mol) of Triton B is added. The deeply coloredsolution is allowed to stand overnight. Water (2 ml) is added. Afterstanding for 15 minutes, the mixture is poured into an excess of water.The organics are extracted with ether (2×50 ml). The aqueous phase isadded to 10% HCl-ice. The orange, gummy solid that precipitates isextracted into methylene chloride and dried (MgSO₄). The solvent isremoved to leave an orange solid. The solid is filtered to give a crudeproduct which is recrystallized to give5,6-difluoro-2-methyl-1-(3,4,5-trimethoxy-benzylidene)-indene-3-aceticacid. When 2,4,6-trimethoxybenzaldehyde or 2,4,5-trimethoxybenzdehyde isused in the above procedure, instead of 3,4,5-trimethoxy-benzaldehyde,the corresponding indene acetic acid is obtained.

(G)5,6-Difluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide

Following the procedure of Example 1(G) and using the acid describedabove leads to the formation of the title compound. (R and R₁ =H, R₂ andR₃ =F, R₄ =H, R₅, R₆ and R₇ =OCH₃, R₈ =CH₃, o=1, M=PhCH₂ NH)

EXAMPLE 685,6-Difluoro-2-Methyl-1-(3,4,5-Trimethoxnbenzylidene)-3-(N-Benzyl)-Indenylacetamide

(A) 3,4-difluorobenzaldehyde.

57 g (0.5 mol) of ortho-difluorobenzene in 250 ml of methylene chlorideis added to 100 g (0.75 mol) of anhydrous aluminum chloride. The mixtureis stirred mechanically at 0° C. (ice bath) while 85.5 g (0.75 mol) ofdichloromethyl methylether is added dropwise. Vigorous HCl evolutiontakes place, and the reaction mixture turns orange-red. After theaddition, the mixture is stirred at room temperature for 15 minutes, andthe liquid phase is decanted into 500 ml of ice and water. The residuein the reaction flask is washed with methylene chloride until colorless,and the washings are added to the water. The mixture is shaken well in aseparatory funnel until the methylene chloride layer is green. Theorganic layer is washed with saturated potassium carbonate solutionuntil neutral, is dried MgSO₄) and is distilled to give3,4-difluorobenzaldehyde, B.P. 70-74° C./20 mm. The dark residue in thedistillation pot solidifies on cooling to givetris-(3,4-difluorophenyl)methane, M.P. 95-96° C.

3,4-DIMETHYLBENZALDEHYDE

The above reaction of Example 68(A) is repeated except that o-xylene anddichloromethyl methylether are the starting materials. Using the samereaction conditions and techniques, there is obtained3,4-dimethylbenzaldehyde.

4-MERCAPTOBENZALDEHYDE

The above reaction of Example 68(A) is repeated except that the startingmaterials are mercaptobenzene and dichloromethyl methylether. Using thesame reaction conditions and techniques, there is obtained4-mercaptobenzaldehyde.

(B)5,6-difluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-Benzyl)-indenylacetamide

The reactions of Examples 67(B)-(G) are repeated and5,6-difluoro-2-methyl-1-(3,4,5-trimethoxy-benzylidene)-3-(N-Benzyl)-indenylacetamideis obtained. (R and R₁ and R₃ =F, R₄ =H, R₅, R₆ and R₇ =OCH₃, R₈ =CH₃,o=1, M=PhCH₂ NH)

Similarly, when 3,4-dimethylbenzaldehyde is used in the reactions inExample 68(B),5,6-dimethyl-2-methyl-1-(3,4,5-trimethoxy-benzylidene)-3-(N-benzyl)-indenylacetamideis obtained. (R₂ and R₃ =CH₃).

When 4-mercaptobenzaldehyde is used in the reactions in Example 68(B),6-mercapto-2-methyl-1-(3,4,5-trimethoxy-benzylidene)-3-(N-benzyl)-indenylacetamideis obtained. (R₂ =H, R₃ =SH, R₄ =H)

EXAMPLE 69α-1-(3,4,5-Trimethoxybenzylidene)-2-Methyl-5-Methoxy-6-Fluoro-3-(N-Benzyl)-Indenylacetamide

(A) 3-fluoro-4-methoxybenzaldehyde.

A solution of o-fluoroanisole, 101 g (0.80 mol) in 500 ml dry methylenechloride is added dropwise over 30 minutes to a solution of titaniumtetrachloride, 182 g (0.96 mol, 1.2 equiv.) anda,(x-dichloromethylmethyl ether, 110 g (0.96 mol) in an equal volume ofmethylene chloride. The temperature is maintained at 10-20° C. with anice-bath. The mixture is stirred at room temperature for 1 hour longerand then poured over crushed ice-water with stirring. Ether (1) isadded, and the mixture stirred under nitrogen until solution occurs. Theorganic layer is extracted with water (3×), with sodium bicarbonatesolution (3×) and is dried (MgSO₄). The solvent is evaporated at 30° C.to give an oily crude product, which is distilled in vacuo through ajacketed Vigreux column to give 3-fluoro-4-methoxybenzaldehyde, B.P.120-121° C. (10 mm Hg) R_(f) 0.6 on a silica-gel G with methylenechloride.

(B) 3-fluoro-4-methoxy-α-methylcinnamic acid.

A mixture of 3-fluoro-4-methoxybenzaldehyde, 34.2 g (0.22 mol),propionic anhydride, 50 g (0.38 mol) and sodium propionate, 21 g (0.22mol) is stirred under nitrogen at 1 50° C. for 15 hours. The reactionmixture is then poured into 1.3 1 of water with stirring, and theproduct is precipitated. 2.0 N potassium hydroxide solution (500 ml) isadded, and the mixture is stirred for several hours, until theprecipitate has dissolved.

The aqueous solution is extracted with ether (3×) and is acidified withconcentrated hydrochloric acid with stirring. The precipitated productis collected, is washed thoroughly with water, and is dried in a vacuumoven at 50° C. over potassium hydroxide pellets to give3-fluoro-u.-methyl-4-methoxycinnamic acid, M.P. 167-169° C.; R_(f) 0.5on silica-gel G with methylene chloride-methanol (1:1).

(C) 3-fluoro-4-methoxy-α-methyl dihydrocinnamic acid.

3-Fluoro-4-methoxy-α-methylcinnamic acid (49.5 g 0.236 mol), in 800 mlmethanol is hydrogenated at 43 lbs. pressure and room temperature untilthe theoretical uptake of hydrogen has occurred (24 min at 20° C., using1.5 g platinum oxide catalyst).

The solution is filtered, and is evaporated with warming to 60° C. togive 3-fluoro-4-methoxy-α-methyl dihydrocinnamic acid (R_(f) 0.5 onsilica-gel G with methylene chloride-methanol, 9:1 )

(D) 5-fluoro-6-methoxy-2-methylindanone.

A mixture of 3-fluoro-α-methyl-4-methoxy dihydrocinnamic acid, 49.3 g(0.23 mol) in 500 g of polyphosphoric acid is heated at 95° C. on asteam bath with occasional agitation for 75 min. The dark red solutionis poured into 3.0 liters of water, and the mixture is stirredovernight. The precipitated product is collected, is washed thoroughlywith water and is dissolved in ether. The ether solution is extractedwith aqueous potassium bicarbonate (4×), diluted with methylenechloride, and dried (MgSO₄).

The organic solution is evaporated and recrystallized from methylenechloride-petroleum ether to give 5-fluoro-6-methoxy-2-methylindanone(M.P. 76-78° C.).

(E) Methyl 6-fluoro-5-methoxy-2-methyl-3-indenylacetate.

Into a 500 ml three-necked flask fitted with mechanical stirrer, refluxcondenser, drying tube, dropping funnel and nitrogen inlet is placed 8.0g zinc sheet and 100 ml of dry benzene. A crystal of iodine is added. Asolution of 21.3 g (0.11 mol) of 5-fluoro-6-methoxy-2-methylindanone and18.36 g (0.121 mol) of methyl bromoacetate in 100 ml of dry benzene isadded in increments of a few milliliters. The mixture is gently heatedwith stirring. After the iodine color has disappeared, and the remainderof the mixture is added gradually, the reaction flask is heated atreflux temperature for about 18 hours The mixture is poured onto 600 mlof 5% H₃ SO₄ and about 500 g of ice. Some ether is added. The organiclayer is separated and washed with three portions of 5% H₂ SO₄ water,KHCO₃ solution and finally water again. The organic layer is dried(MgSO₄) and concentrated to give 27.6 g of reddish oil whichcrystallizes upon standing. Thin-layer chromatography on silica-gel Gwith methylene chloride methanol (99:1 ) shows product at R_(f) (0.5).

Without further purification, the hydroxy ester is dehydrated to theindenylacetate. In 200 ml of dry benzene, 14.2 g (53 mol) of crude esterand 36 g of phosphorus pentoxide are refluxed with stirring for 1/2hour. After cooling, the reaction mixture is filtered and the solidresidue washed well with benzene. The benzene filtrate is washed withtwo portions of salt water and dried (MgSO₄). The organic solution isconcentrated and gives a slightly colored oil which rapidlycrystallizes. The crude product is recrystallized from methylenechloride-petroleum ether to givemethyl-6-fluoro-5-methoxy-2-methyl-3-indenylacetate (M.P. 61-62° C.).

(F)6-fluoro-5-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylacetic acid.

To a solution of methyl-6-fluoro-5-methoxy-2-methyl-3-indenyl acetate,9.3 g (0.037 mol) and 3,4,5-trimethoxy-benzaldehyde, 7.99 g (1.1equivalent) is added 16 ml (2.0 equivalents) of 25% methanolic sodiummethoxide. The mixture is stirred at reflux under nitrogen for 2 hours.An equal volume of water is added dropwise and refluxing continues for30 minutes. The solution is cooled, diluted with water and extractedwith ether (3×). Residual ether is blown off with nitrogen, and then theaqueous solution is acidified with 50% glacial acetic acid. Theprecipitated product is collected and washed thoroughly with water. Thecrude product is recrystallized to give6-fluoro-5-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylacetic acid.

(G)6-fluoro-5-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide

Following the procedures of Example 1(F) and (G) and using the aciddescribed above leads to the formation of the title compound. (R and R₁=H, R₂ =OCH₃, R₃ =F, R₄ =H, R₅, R₆ and R₇ =OCH₃, R₈ =CH₃, o=1, M=PhCH₂NH)

EXAMPLE 70Cis-5,7-Difluoro-2-Methyl-1-(3,4,5-Trimethoxybenzylidene)-3-(N-Benzyl)-IndenylAcetamide

(A) 2,4-difluorobenzaldehyde.

A 250 ml, three-necked flask is fitted with a stirrer, a thermometer, adropping funnel with a long stem to the bottom of the flask and a refluxcondenser with a tube leading to the back of a hood. 50 g (0.38 mol) of2,4-difluorotoluene is heated to reflux with stirring and irradiatedwith a Hanovia ultraviolet lamp. 41.5 ml of bromine is gradually added.The reaction is completed in 2.5 hours during which time the refluxtemperature rises from 112° C. to 155° C.

A 2 l three-necked flask is fitted with a stirrer and reflux condenser.In the flask is placed 200 ml of water and 140 g calcium carbonate. Thecooled above-described reaction mixture is transferred into the 21three-necked flask using some ether for rinsing. The hydrolysis iscompleted by refluxing the reaction mixture with stirring for 18 hours.The aldehyde is isolated by steam distillation. The oil is separated andthe aqueous phase is extracted once with ether. The oil and etherextracts are combined and dried over anhydrous MgSO₄ and concentratedunder reduced pressure. The obtained 2,4-difluorobenzaldehyde, containssome ether and is purified by distillation over a short Vigreux columnunder reduced pressure. B.P. 56-58° C. 12 mm.

(B) 2,4-difluoro-ox-methylcinnamic acid.

A 500 ml, three-necked flask is fitted with a reflux condenser, dryingtube, stirrer and N₂ inlet. To a mixture of 55.4 g (0.39 mol) of2,4-difluorobenzaldehyde and 56 ml of propionic anhydride is added 38 g(0.39 mol) of sodium propionate. The reaction mixture is heated at135-140° C. (oil bath temp.) for 19 hours with stirring under nitrogen.The still warm solution is poured into 1 l of water with stirring. Asolid separates, which is dissolved by 56 g of potassium hydroxide. Thesolution is extracted with ether. The water phase is heated on the steambath to remove the ether. After cooling in an ice-bath, concentratedhydrochloric acid is added with stirring. The solid which separates iscollected and washed with cold water. Drying at 60° C. over KOH gives2,4-difluoro-α-methylcinnamic acid, M.P. 126-128° C.

(C) 2,4-difluoro-α-methyldihydrocinnamic acid.

In 800 ml of methanol, 60 g (0.3 mol) of 2,4-difluoro-α-methylcinnamicacid with 1.5 g of platinum oxide catalyst is shaken under an initialpressure of 42 lbs of hydrogen until one equivalent of hydrogen isabsorbed. The reaction time is 30 minutes. The catalyst is removed byfiltration and washed with methanol. The methanol is evaporated off. Theresidue, a near-colorless oil, 2,4-difluoro-α-methyldihydrocinnamicacid, is used in the next step without further purification.

(D) 4,6-difluoro-2-methylindanone.

A solution of 2,4-difluoro-α-methyldihydrocinnamic acid, 54.8 g (0.274mol) in 125 ml thionyl chloride is stirred for 90 minutes, at roomtemperature, and then refluxed for 90 minutes. The thionyl chloride isevaporated under reduced pressure. The acid chloride is obtained as anoil.

To a suspension of powdered aluminum chloride, 60 g (0.45 mol), in 250ml of dry carbon disulfide at 0° C. is added dropwise over a period of10 minutes, a solution of the acid chloride, 60 g, in 100 ml carbondisulfide. After the addition, the ice bath is removed, and thetemperature raised slowly to room temperature. The mixture is stirred atroom temperature for 20 hours, and then is poured into 2 l of 10%aqueous hydrochloric acid-crushed ice with stirring. Ether is added, andthe stirring is continued until everything dissolves. The ether layer isseparated, is extracted with 5% hydrochloric acid (2×), water (2×), andsodium bicarbonate solution (2×), and is dried (MgSO₄). The ether isevaporated to give the crude 4,6-difluoro-ot-methylindanone as an oilwhich crystallizes on standing. The crude product is purified by flashchromatography on silica-gel, 400 g of J. T. Baker 3405 with petroleumether-methylene chloride (2:1). Recrystallization from methylenechloride-petroleum ether gives 4,6-difluoro-2-methylindanone, M.P.68-69° C.

(E) Methyl 5,7-difluoro-2-methylindenyl-3-acetate.

About 20% of a solution containing 4,6-difluoro-2-methylindanone, 15.0 g(83 mmol), and methyl bromoacetate, 14.0 g (1.1 equiv.) in 100 ml drybenzene is added to a stirred suspension of powdered zinc dust (Merckdried 120° C./22 mm.), 6.5 g (1.2 equiv.) in 74 ml dry benzene under anitrogen atmosphere. Several crystals of iodine are added, and themixture slowly brought to a reflux. The remainder of the solution isadded dropwise over 10 minutes, and the mixture stirred at refluxovernight, i.e., 17 hours. The reaction mixture is cooled to roomtemperature, and is poured into 2.0 1 of 20% aqueous sulfuricacid-crushed ice with stirring. Ether is added until a clear solution isobtained. The ether layer is extracted with 5% aqueous sulfuric acid(3×), water (3×), diluted with methylene chloride and dried (MgSO₄). Thesolvents are evaporated to give crude hydroxy ester.

Powdered phosphorus pentoxide (60.0 g) is added to a solution of thehydroxy ester (20.0 g) in 400 ml of dry benzene. The mixture is refluxedand stirred for 30 minutes. The clear benzene solution is decanted. Theresidue is rinsed with benzene and then with ether. The combined organicsolutions are diluted with ether, extracted six times with aqueoussodium sulfate solution, twice with aqueous potassium bicarbonatesolution, diluted with methylene chloride and dried (MgSO₄). The crudeindenyl acetate product is obtained as an oil when the solvents areevaporated. The product is crystallized from petroleum ether to givemethyl 5,7-difluoro-2-methylindenyl-3-acetate, M.P. 69-70° C.

(F) 5,7-difluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylacetic acid, a mixture of geometric isomers.

Powdered sodium methoxide, 2.2 g (40 mmol) is added to a suspension ofmethyl 5,7-difluoro-2-methyl-indenyl-3-acetate (4.78 g) (20 mmol) and3,4,5-trimethoxybenzaldehyde, 4.32 g (22 mmol), in 40 ml dry methanolunder nitrogen. A clear solution results which is refluxed for 60minutes. An equal volume of water is added, and refluxing continuedunder nitrogen for 30 minutes to complete saponification. The coldsolution is diluted with water and extracted with ether. Nitrogen isbubbled through the aqueous solution to remove the residual ethersolvent. Fifty percent aqueous acetic acid (40 ml) is used toprecipitate the product. The product is filtered off and washed withwater. It is dried in a desiccator over potassium hydroxide pellets, andfinally in the oven at 100° C. The crude product is recrystallized togive a mixture of the cis and trans isomers of the acid.

(G)Cis-methyl-5,7-difluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenyl-acetateisolation by column chromatography.

Four drops of concentrated sulfuric acid are added to a solution of5,7-difluoro-2-methyl-1-(3,4,5-trimethoxy-benzylidene)-3-indenyl aceticacid, 1.0 g (2.8 mmol) in 60 ml of dry methanol, and the solution isstirred at reflux overnight. The solution is cooled and crystalsseparated. They are collected, rinsed with cold methanol-water (1:1) anddried over potassium hydroxide pellets. These crystals are found to beabout 95% of the trans-isomer, and could be further purified byrecrystallization to give the pure trans-isomer. Water is added to thefiltrate from the first crop and a second crop of crystals is obtained.The second crop of crystals is cis-enriched and used for chromatographyafter it was allowed to react with diazomethane.

1.7 g of cis and trans-mixed esters are chromatographed on a column(3.0×90 cm) of silica-gel, 250 g of J. T. Baker 3405. The column isdeveloped and eluted with halogenated solvents. In this way thetrans-isomer and the cis-isomer may be obtained.

(H) Cis-5,7-difluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylacetic acid.

0.1 N aqueous sodium hydroxide 3.0 ml (3.0 mmol) is added to cis-methyl5,7-difluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylacetate,266 mg (0.64 mmol) in 20 ml methanol under nitrogen. The mixture isrefluxed for 1 hour, cooled, diluted with water and acidified withseveral ml of 50% acetic acid Crystals form and after further chillingin ice bath, they are collected, washed thoroughly with water and suckednearly dry. The product is recrystallized, dried over potassiumhydroxide pellets in a vacuum desiccator and finally in a vacuum oven at100C. In this waycis-5,7-difluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylacetic acid may be obtained.

(I)cis-5,7-difluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide

Following the procedures of Example 1(F) and (G) and using the aciddescribed above leads to the formation of the title compound. (R and R₁=H, R₂ =F, R₃ =H, R₄ =F, R₅, R₆ and R₇ =OCH₃, R₈ =CH₃, o=1, M=PhCH₂ NH)

EXAMPLE 71α-(1-(3,4,5-Trimethoxybenzylidene)-2-Methyl-5,6-Difluoro-3-Indenyl)-(N-Benzyl)-Propionamide

α-[1-(3,4,5-trimethoxybenzylidene)-2-methyl-5,6-difluoro-3-indenyl]-(N-benzyl)-propionamideis prepared following the procedures of Examples 64(A)-(C). (R=H, R₁=CH₃, R₂ =H, R₃ =F, R₄ =F, R₅ =R₆ =R₇ =OCH₃, R₈ =CH₃, o=1, M=PhCH₂ NH)

EXAMPLE 72α-(1-(3,4-Trimethoxybenzylidene)-2-Methyl-5-Fluoro-6-Methoxy-3-Indenyl)-(N-Benzyl)-Propionamide

α-[1-(3,4,5-trimethoxybenzylidene)-2-methyl-5-fluoro-6-methoxy-3-indenyl]-(N-benzyl)-propionamideis prepared following the procedures of Examples 64(A)-(C). (R=H, R₁=CH₃, R₂ =H, R₃ =F, R₄ =OCH₃, R₅ =R₆ =R₇ =OCH₃, R₈ =CH₃, o=1, M=PhCH₂NH)

EXAMPLE 73α-(1-(2,4,6-Trimethoxybenzylidene-2-Methyl-5-Fluoro-3-Indenyl)-(N-Benzyl)-Propionamide

α-[1-(2,4,6-trimethoxybenzylidene)-2-methyl-5-fluoro-3-indenyl]-(N-benzyl)-propionamideis prepared following the procedures of Examples 64(A)-(C). (R=H, R₁=CH₃, R₂ =H, R₃ =F, R₄ =H, R₅ =R=R₇ =OCH₃, R₈ =CH₃, o=1, M=PhCH₂ NH)

EXAMPLE 74(Z)-2-Methyl-1-(3,4,5-Trimethoxybenzylidene-3-(N-Benzyl)-Indenylformamide

Following the procedures of Example 1(E)-(G) and using3-methylindene-2-carboxylic acid [Beil. 9, 644; Aldrich] leads to theformation of the title compound. (R=R₁ =do not exist, because o=0; R₂=H, R₃ =H, R₄ =H, R₅ =R₆ =R₇ =OCH₃, R₈ =CH₃, o=0, M=PhCH₂ NH)

EXAMPLE 75(Z)-5-Fluoro-2-Methyl-1-(3,4,5-Trimethoxybenzylidene)-3-(N-Benzyl)-Indenylpropionamide

(A)(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylmethyldiazomethyl ketone

Diazomethane (excess) in ether (700 ml) is allowed to react with theacid chloride (Example 1(F), 5 g, 12.5 mmol) in CH₂ Cl₂ (100 ml). After30 min, the solvent was evaporated to give the title compound, which wasused in the next step without purification.

(B)(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylpropionicacid

Sodium hydroxide (7 ml, 1M) is added to a solution of silver nitrate (1g, 5.89 mmol) in water (9 ml). The silver oxide is filtered and isstirred with a solution of (Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylmethyl diazomethyl ketone (1 g, 2.44mmol) in ethanol (100 ml) for 3 h under reflux conditions. The solutionis filtered. The filtrate is treated with base (2N NaOH, 1 d) andacidified with 2N HCl. The product is extracted with ethyl acetate. Theorganic phase is washed with water and is dried over Na₂ SO₄.Recrystallization from acetonitrile gives the title compound.

(C)(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylpropionamide

Following the procedure of Example 1(F) and 1(G) and using(Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylpropionicacid instead of(Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylaceticacid leads to the formation of the title compound. (R=H, R₁ =H, R₂ =F,R₃ =H, R₄ =H, R₅ =R₆ =R₇ =OCH₃, R₁ =CH₃, o=2, M=PhCH₂ NH)

EXAMPLE 76(Z)-5-Fluoro-2-Methyl-1-(3,4,5-Trihydroxybenzylidene)-3-(N-Benzyl)-Indenylacetamide

BBr₃ (4.72 ml, 50 mmol) in CH₂ Cl₂ (30 ml) is added slowly at -78° C. toa solution of(Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide(Example 1(G), 5 g, 10.56 ml) in CH₂ Cl₂ (150 ml). The reaction mixtureis stirred at -78° C. for 2 h, then at 25° C. for two days. Aftercooling, the reaction mixture is poured into the ice-water (200 ml), andis extracted with ethyl acetate. The organic phase is dried (Na₂ SO₄)and is evaporated. The solution of the residue in CH₃ OH: H₂ C) (7:3) ischromatographed on reverse phase silica gel to give(Z)-5-fluoro-2-methyl-1-(3,4,5-trihydroxybenzylidene)-3-(N-benzyl)-idenylacetamide.

(R=H, R₁ =H, R₂ =F, R₃ =H, R₄ =H, R₅ =R₆ =R₇ =OH, R₈ =CH₃, o=1, M=PhCH₂NH).

BIOLOGICAL EFFECTS

(A) Growth Inhibition

These compounds were assayed for their growth inhibitory activity on thehuman colon carcinoma cell line, SW-480 obtained from ATCC (Rockville,Md.), to ascertain the degree of growth inhibition. Growth inhibition ofthis cell line is indicative of a benefit on precancerous lesions andneoplasms. The cell line and growth assay employed for these experimentsare well characterized, and are used to evaluate the anti-neoplasticproperties of NSAIDs. The assay is used by the United States NationalCancer Institute in its screening program for new anti-cancer drugs.

Drug stock solutions were made in 100% DMSO then diluted with RPMI mediafor cell culture testing. All drug solutions were prepared fresh on theday of testing. The cultured cells were obtained at passage #99 andgrown in RPMI media supplemented with 5% fetal calf serum, and 2 mMglutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 0.25 1μg/mlamphotericin. The cultures were maintained in a humidified atmosphere of95% air and 5% CO₂ at 37° C. The cultures were passaged at preconfluentdensities using a solution of 0.05% trypsin and 0.53 mM EDTA. Cells wereplated at 1000 cells/well for 96 well flat-bottom microtiter plates.

Tumor cell growth inhibition was assessed using the Sulforhodamine B(SRB) protein binding assay. In this assay, tumor cells were plated in96-well plates and treated with drug-containing media for six days(continuous exposure). For each plate, 6 wells were designated as notreatment controls, six wells as vehicle (0.1% DMSO) controls, and theremaining wells for drug dilutions with three wells per drugconcentration. At the end of the exposure period, the cells were fixedand stained with sulforhodamine B, a protein binding dye. The dye wasthen solubilized, and the optical density of the resulting solution wasdetermined on a 96-well plate reader. The mean dye intensity of thetreated wells was then divided by the mean dye intensity in the controlwells (6 wells of each) to determine the effect of the drug on thecells. Dye intensity is proportional to the number of cells or amount ofprotein per well. The resultant "percent inhibition" value thenrepresents the degree of growth inhibition caused by the drug.

For each experiment, an IC₅₀ value was determined and used forcomparative purposes. This value is equivalent to the concentration ofdrug needed to inhibit tumor cell growth by 50%. IC₅₀ values wereobtained graphically by connecting the mean values for each drugconcentration tested. Each experiment included at least three wells perdrug concentration. Concentration was plotted on a log scale on theX-axis. IC₅₀ values obtained for the compounds of different Examples areprovided in Table 4 below for the SW-480 cell line.

                  TABLE 4                                                         ______________________________________                                               EXAMPLE IC.sub.50 (μM)                                              ______________________________________                                               1       0.04                                                             4 0.36                                                                        8 0.12                                                                        15 0.39                                                                       19 4.5                                                                        22 4.5                                                                        23 8.0                                                                        27 0.22                                                                       28 0.37                                                                       31 5.2                                                                        32 2.2                                                                        33 1.8                                                                        34 6.2                                                                        35 >10                                                                        36 3                                                                          37 >10                                                                        38 >10                                                                        39 >10                                                                        40 >10                                                                        41 >10                                                                        42 0.69                                                                       43 >10                                                                        44 >10                                                                        45 >10                                                                        46 >10                                                                        47 4.1                                                                        48 0.5                                                                        51 2.25                                                                       55 0.5                                                                        57 0.26                                                                       58 0.98                                                                     ______________________________________                                    

(B) Cyclooxygenase (COX) Inhibition

Compounds of this invention, as well as a positive control, sulindacsulfidewere evaluated to determine whether they inhibited the productionof cyclooxygenase (see Table 5 below).

COX catalyzes the formation of prostaglandins and thromboxane by theoxidative metabolism of arachidonic acid. The compounds of thisinvention were evaluated for inhibitory effects on purified COX The COXwas purified from ram seminal vescicles, as described by Boopathy, R.and Balasubramanian, J., 239:371-377, 1988. COX activity was assayed asdescribed by Evans, A. T., et al., "Actions of cannabis constituents onenzymes of arachidonate metabolism anti-inflammatory potential, "Biochem. Pharmacol., 36:2035-2037, 1987. Briefly, purified COX wasincubated with achidonic acid (100 μM) for 2.0 min at 37° C. in thepresence or absence of test compounds. The assay was terminated by theaddition of TCA, and COX activity was determined by absorbance at 530nm.

                  TABLE 5                                                         ______________________________________                                                      COX I                                                              % Inhibition (100 μM)                                                     EXAMPLE (* - 1000 μm)                                                    ______________________________________                                        sulindac sulfide                                                                             86                                                               1 <25                                                                         2 <25                                                                         3 <25                                                                         4 <25                                                                         5 <25                                                                         6 <25                                                                         7 <25                                                                         8 <25                                                                         9 <25                                                                         10 <25                                                                        11 <25                                                                        12 <25                                                                        13 <25                                                                        14 <25                                                                        15 <25                                                                        16 <25                                                                        17 <25                                                                        18 <25                                                                        19   33.3                                                                     20   36.5                                                                     21   36.5                                                                     22 <25                                                                        23 <25                                                                        24 <25                                                                        25  25                                                                        26   28.1                                                                     27 <25                                                                        28 <25                                                                        29 <25                                                                        30 <25                                                                        31  30*                                                                       32  58*                                                                       33  49*                                                                       34  31*                                                                       35  37*                                                                       36  72*                                                                       37  38*                                                                       38  35*                                                                       39  59*                                                                       40  34*                                                                       41  59*                                                                       42  49*                                                                       43  36*                                                                       44  51*                                                                       45  56*                                                                       46  98*                                                                       47  37*                                                                       48 <25                                                                        49  25                                                                        50   29.2                                                                     51  <25*                                                                      52 <25                                                                        55 <25                                                                        57 <25                                                                        58 <25                                                                      ______________________________________                                    

(C) Apoptosis

Apoptosis was measured using an assay of cell death based onmorphological characteristics of apoptotic cells (i.e., condensedchromatin). Drug preparation and cell culture conditions were the sameas for the SRB assay described above, except that HT-29 human coloncarcinoma cells were employed. Confluent cultures were established in12.5 cm² flasks by plating 0.5×10⁶ cells/flask. The cultures wereassayed for apoptosis by fluorescent microscopy following labeling withacridine orange and ethidium bromide. Floating and attached cells werecollected by trypsinization and washed three times in PBS. One mlaliquots were centrifuged (3 g). The pellet was resuspended in 25 illmedia and 1 μl of a dye mixture containing 100 μg/ml acridine orange and100 μg/ml ethidium bromide prepared in PBS and mixed gently. Ten μl ofmixture was placed on a microscope slide and covered with a 22 mm²coverslip and examined under 40× dry objectives using epillumination andfilter combination.

An observer blinded to the identity of the treatments scored at least100 cells per sample. Apoptotic cells were identified by nuclearcondensation of chromatin stained by the acridine orange or ethidiumbromide. These results are provided in Table 6 below.

                  TABLE 6                                                         ______________________________________                                        Apoptosis Effects of Compounds                                                              Morphology                                                         % Apoptotic Cells DNA Fragmentation                                        EXAMPLE   (1 μM)      FS (100 μM)                                                                          EC.sub.50 (μM)                          ______________________________________                                        1         73             4.6       0.05                                         4 68 2.4 0.25                                                                 8 48 <2 0.21                                                                  15 72 2.1 0.3                                                                 19 <20 2 7                                                                    22 37 2.3 5                                                                   27 71 4 0.7                                                                   28 35 2.3 0.23                                                                31 30 <2 >10                                                                  32 55 2.5 0.32                                                                33 28 3.7 4                                                                   34 45 2.1 >10                                                                 35 40 <2 >10                                                                  36 22 5.3 0.01                                                                37 <20 2.5 >10                                                                38 20 <2 >10                                                                  39 31 2 >10                                                                   40 <20 <2 >10                                                                 41 30 <2 >10                                                                  42 <20 3.8 3                                                                  43 25 <2 >10                                                                  44 25 2.7 >10                                                                 45 40 3.4 >10                                                                 46 20 4.8 >10                                                                 47 20 4.4 5.7                                                                 48 63 2.5 0.5                                                                 51 40 <2 >10                                                                  57 73 <2 0.3                                                                  58 52 2.5 0.7                                                               ______________________________________                                    

Apoptosis was also measured based on the amount of fragmented DNAcontained in cell lysates. Briefly, SW-480 colon adenocarcinoma cellswere plated in 96-well microtitre plates ("MTP") at a density of 10Kcells/well in 180 μl and incubated for 24 hrs. Cells were then treatedwith 20μl aliquots of appropriately diluted compound, and allowed toincubate for an additional 48 hrs.

After the incubation, samples were prepared according to the followingsteps. The MTP was centrifuged (15 min., 1000 rpm) and the supernatantwas carefully removed by fast inversion of the MTP. The cell pellets ineach well were then resuspended in 200 μl lysis buffer and incubated for45 min. at room temperature to lyse the cells. The lysates were thencentrifuged (15 min., 1000 rpm) and 20 μl aliquots of the supernatant(=cytoplasmic fraction) were transferred into the streptavidin coatedMTP for analysis. Care was taken not to shake the lysed pellets in theMTP (=cell nucleii containing high molecular weight, unfragmented DNA).Samples were analyzed immediately, because storage at 4 C or -20 Creduces the ELISA-signals. Samples were then processed according to aDNA fragmentation assay protocol, and dose-response curves weregenerated based on optical density readings. Quantification of DNA wasdone by a commercially available photometric enzyme-immunoassaymanufactured by Mannheim-Boehringer under the name "Cell Death DetectionELISA ^(plus) ". The assay is based on a quantitativesandwich-enzyme-immunoassay-principle using mouse monoclonal antibodiesdirected against DNA and histones, respectively This allows the specificdetermination of mono and oligonucleosomes in the cytoplasmatic fractionof cell lysates. In brief, the assay procedure is as follows. The sample(cell-lysate, serum, culture-supernatant etc.) is placed into astreptavidin-coated MTP. Subsequently, a mixture of anti-histone-biotinand anti-DNA-POD are added and incubated for 2 hours. During theincubation period, the anti-histone antibody binds to thehistone-component of the nucleosomes and simultaneously fixes theimmunocomplex to the streptavidin-coated MTP via its biotinylation.Additionally, the anti-DNA-POD antibody reacts with the DNA component ofthe nucleosomes. After removal of unbound antibodies by a washing step,the amount of nucleosomes is quantified by the POD retained in theimmunocomplex. POD is determined photometrically withABTS®(2,2'-Azino-di[3-ethyl benzthiazol in-sulfonat])* as substrate.

Fold stimulation (FS=ODmax/ODveh), an indicator of apoptotic response,was determined for each compound tested. EC₅₀ values were determinedeither specifically by data analysis software, or by estimates based onthe effective concentration range of each compound (ECR=min. effectivedose-min. dose to peak effect). These FS and EC₅₀ values for the testedcompounds are listed above in Table 6.

The compounds of this invention can be formulated with pharmaceuticallyacceptable carriers into unit dosage forms in a conventional manner sothat the patient in need of therapy for precancerous lesions canperiodically (e.g., once or more per day) take a compound according tothe method of this invention. The exact initial dose of the compounds ofthis invention can be determined with reasonable experimentation. Oneskilled in the art should understand that the initial dosage should besufficient to achieve a blood plasma concentration approaching apercentage of the IC₅₀ value of the compound, with the percentagedepending on the chemopreventative or chemotherapeutic indication. Theinitial dosage calculation would also take into consideration severalfactors, such as the formulation and mode of administration, e.g. oralor intravenous, of the particular compound. For example, assuming apatient with an average circulatory system volume of about four liters,then based on the reported IC₅₀ value for the compound of Example No. 1,one would calculate a dosage of about 0.075 mg of this compound forintravenous administration to achieve a systemic circulatoryconcentration equivalent to the IC₅₀ concentration.

It will be understood that various changes and modifications can be madein the details of procedure, formulation and use without departing fromthe spirit of the invention, especially as defined in the followingclaims.

We claim:
 1. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and a compound of formula I or pharmaceuticallyacceptable salt thereof: ##STR2## wherein R and R₁ are independentlyselected from the group consisting of hydrogen, hydroxy, lower alkyl andamino; or R and R₁ together may be oxygen;R₂, R₃ and R₄ areindependently selected from the group consisting of hydrogen, hydroxy,halogen, lower alkoxy, lower alkyl and alkyl mercapto; at least two ofR₅, R₆ and R₇ are identically selected from the group consisting ofhydroxy and lower alkoxy, and the third is selected from the groupconsisting of hydroxy, halogen, lower alkoxy, lower alkyl, amino andlower dialkylamino; with the proviso that when at least one of R₂, R₃ orR₄ is lower alkoxy, then each of R₅, R₆ and R₇ are hydroxy or loweralkoxy; R₈ is selected from the group consisting of hydrogen and loweralkyl; o is 0, 1 or 2; and M is selected from the group consisting ofamino, alkylamino, dialkylamino, alkoxyamino, alkenylamino,alkynylamino, hydroxyalkylamino, polyhydroxyalkyl-amino,dialkylaminoalkylamino, aminoalkylamino, benzylamino, anilino,phenylalkylamino, aminoindan, and heterocycloalkylamino where theheterocycles are selected from the group consisting of pyridinyl,piperidinyl, piperazinyl, pyrrollidinyl and N-morpholino; wherein thealkyl between the cyclic structure and the amino may be absent, and thecyclic structures may optionally be substituted with one or more ofhalo, alkoxy, hydroxy, amino, alkylamino, dialkylamino and sulfonamido.2. The pharmaceutical composition of claim 1 whereino is 1 or 2; and Mis selected from the group consisting of amino, alkylamino,dialkylamino, alkenylamino, alkynylamino, hydroxyalkylamino,dialkylaminoalkylamino, arylalkylamino selected from the groupconsisting of benzylamino, anilino and phenylalkylamino, aminoindan, andheterocycloalkylamino where the heterocycles are selected from the groupconsisting of pyridinyl, piperidinyl, piperazinyl, and pyrrollidinyl;wherein the alkyl between the cyclic structure and the amino may beabsent, and the cyclic structures may optionally be substituted with oneor more of halo, alkoxy, hydroxy, amino, alkylamino and dialkylamino. 3.The pharmaceutical composition of claim 2 wherein M is selected from thegroup consisting of alkenylamino, alkynylamino, benzylamino, andpyridinylalkylamino; wherein the cyclic structures may optionally besubstituted with one or more of halo, alkoxy, hydroxy, amino, alkylaminoand dialkylamino.
 4. The pharmaceutical composition of claim 3 wherein Mis selected from the group consisting of alkenylamino, alkynylamino,benzylamino, and pyridinylalkylamino; wherein the cyclic structures mayoptionally be substituted with one or more of halo, alkoxy, hydroxy,amino, alkylamino and dialkylamino.
 5. The pharmaceutical composition ofclaim 4 wherein M is selected from benzylamino and benzylaminosubstituted with one or more of halo, alkoxy, hydroxy, amino, alkylaminoand dialkylamino.
 6. The pharmaceutical composition of claim 4 whereinRand R₁ are independently selected from the group consisting of hydrogenand hydroxy; and at least two of R₅, R₆ and R₇ are lower alkoxy and thethird is selected from hydroxy, lower alkoxy, amino and lowerdialkylamino; or each of R₅, R₆ and R₇ are hydroxy.
 7. Thepharmaceutical composition of claim 6 whereinR and R₁ are hydrogen; R₂,R₃ and R₄ are independently selected from the group consisting ofhydrogen, hydroxy, halogen, lower alkoxy, lower alkyl and alkylmercapto; at least two of R₅, R₆ and R₇ are lower alkoxy and the thirdis selected from hydroxy and lower alkoxy; R₈ is lower alkyl, o is 1;and M is selected from the group consisting of alkenylamino,alkynylamino, benzylamino, and pyridinylalkylamino; wherein the cyclicstructures may optionally be substituted with one or more of halo,alkoxy, hydroxy, amino, alkylamino and dialkyl amino.
 8. Thepharmaceutical composition of claim 7 whereinR₅, R₆ and R₇ are eachlower alkoxy; and R₈ is methyl.
 9. The pharmaceutical composition ofclaim 8 whereinR₂ is selected from the group consisting of hydroxy,halogen, lower alkoxy and alkyl mercapto; R₄ is hydrogen; and R₅, R₆ andR₇ are each methoxy.
 10. The pharmaceutical composition of claim 9whereinR₂ is selected from the group consisting of hydroxy, halogen andlower alkoxy; and R₃ is hydrogen.
 11. The pharmaceutical composition ofclaim 10 wherein R₂ is halogen.
 12. The pharmaceutical composition ofclaim 11 wherein R₂ is fluoro.
 13. The pharmaceutical composition ofclaim 12 wherein the compound is selected from the group consistingof:(Z)-5-Fluoro-2-methyl-1-(2,4,6-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide;(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide;(E)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide;(Z)-5-Fluoro-2-methyl-1-(2,3,4-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide;and(Z)-5-Fluoro-2-methyl-1-(2,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide.14. The pharmaceutical composition of claim 13 wherein the compoundis(Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide.15. A method of treating a patient having precancerous lesionscomprising administering a pharmacologically effective amount of acompound of formula I or pharmaceutically acceptable salt thereof:##STR3## wherein R and R₁ are independently selected from the groupconsisting of hydrogen, hydroxy, lower alkyl and amino; or R and R₁together may be oxygen;R₂, R₃ and R₄ are independently selected from thegroup consisting of hydrogen, hydroxy, halogen, lower alkoxy, loweralkyl and alkyl mercapto; at least two of R₅, R₆ and R₇ are identicallyselected from the group consisting of hydroxy and lower alkoxy, and thethird is selected from the group consisting of hydrogen, hydroxy,halogen, lower alkoxy, lower alkyl, amino and lower dialkylamino; withthe proviso that when at least one of R₂, R₃ or R₄ is lower alkoxy, theneach of R₅, R₆ and R₇ are hydroxy or lower alkoxy; R₈ is selected fromthe group consisting of hydrogen and lower alkyl; o is 0, 1 or 2; and Mis selected from the group consisting of amino, alkylamino,dialkylamino, alkoxyamino, alkenylamino, alkynylamino,hydroxyalkylamino, polyhydroxyalkyl-amino, dialkylaminoalkylamino,aminoalkylamino, benzylamino, anilino, phenylalkylamino, aminoindan, andheterocycloalkylamino where the heterocycles are selected from the groupconsisting of pyridinyl, piperidinyl, piperazinyl, pyrrollidinyl andN-morpholino; wherein the alkyl between the cyclic structure and theamino may be absent, and the cyclic structures may optionally besubstituted with one or more of halo, alkoxy, hydroxy, amino, alkylamino, dialkylamino and sulfonamido.
 16. The method of claim 15 whereinois 1 or 2; and M is selected from the group consisting of alkenylamino,alkynylamino, benzylamino, and pyridinylalkylamino; wherein the cyclicstructures may optionally be substituted with one or more of halo,alkoxy, hydroxy, amino, alkylamino and dialkylamino.
 17. The method ofclaim 16 wherein M is selected from the group consisting ofalkenylamino, alkynylamino, benzylamino, and pyridinylalkylamino;wherein the cyclic structures may optionally be substituted with one ormore of halo, alkoxy, hydroxy, amino, alkylamino and dialkylamino. 18.The method of claim 17 wherein M is selected from benzylamino andbenzylamino substituted with one or more of halo, alkoxy, hydroxy,amino, alkylamino and dialkylamino.
 19. The method of claim 17 whereinRand R₁ are independently selected from the group consisting of hydrogenand hydroxy; and at least two of R₅, R₆ and R₇ are lower alkoxy and thethird is selected from hydroxy, lower alkoxy, amino and lowerdialkylamino; or each of R₅, R₆ and R₇ are hydroxy.
 20. The method ofclaim 19 whereinR and R₁ are hydrogen, R₂, R₃ and R₄ are independentlyselected from the group consisting of hydrogen, hydroxy, halogen, loweralkoxy, lower alkyl and alkyl mercapto; at least two of R₅, R₆ and R₇are lower alkoxy and the third is selected from hydroxy and loweralkoxy; R₈ is lower alkyl; o is 1; and M is selected from the groupconsisting of alkenylamino, alkynylamino, benzylamino, andpyridinylalkylamino, wherein the cyclic structures may optionally besubstituted with one or more of halo, alkoxy, hydroxy, amino, alkylaminoand dialkylamino.
 21. The method of claim 20 whereinR₅, R₆ and R₇ areeach lower alkoxy; and R₈ is methyl.
 22. The method of claim 21whereinR₂ is selected from the group consisting of hydroxy, halogen,lower alkoxy and alkyl mercapto; R₄ is hydrogen; and R₅, R₆ and R₇ areeach methoxy.
 23. The method of claim 22 whereinR₂ is selected from thegroup consisting of hydroxy, halogen and lower alkoxy; and R₃ ishydrogen.
 24. The method of claim 23 wherein R₂ is fluoro.
 25. Themethod of claim 24 wherein the compound is selected from the groupconsistingof:(Z)-5-Fluoro-2-methyl-1-(2,4,6-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide;(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide;(Z)-5-Fluoro-2-methyl-1-(2,3,4-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide;and(Z)-5-Fluoro-2-methyl-1-(2,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide.26. The method of claim 25 wherein the compound is(Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide.27. A method for inhibiting the growth of neoplastic cells comprisingexposing the cells to a growth inhibiting effective amount of a compoundof formula I or pharmaceutically acceptable salt thereof: ##STR4##wherein R and R₁ are independently selected from the group consisting ofhydrogen, hydroxy, lower alkyl and amino; or R and R₁ together may beoxygen;R₂, R₃ and R₄ are independently selected from the groupconsisting of hydrogen, hydroxy, halogen, lower alkoxy, lower alkyl andalkyl mercapto; at least two of R₅, R₆ and R₇ are identically selectedfrom the group consisting of hydroxy and lower alkoxy, and the third isselected from the group consisting of hydrogen, hydroxy, halogen, loweralkoxy, lower alkyl, amino and lower dialkylamino; with the proviso thatwhen at least one of R₂, R₃ or R₄ is lower alkoxy, then each of R₅, R₆and R₇ are hydroxy or lower alkoxy; R₈ is selected from the groupconsisting of hydrogen and lower alkyl; o is 0, 1 or 2; and M isselected from the group consisting of amino, alkylamino, dialkylamino,alkoxyamino, alkenylamino, alkynylamino, hydroxyalkylamino,polyhydroxyalkyl-amino, dialkylaminoalkylamino, aminoalkylamino,benzylamino, anilino, phenylalkylamino, aminoindan, andheterocycloalkylamino where the heterocycles arc selected from the groupconsisting of pyridinyl, piperidinyl, piperazinyl, pyrrollidinyl andN-morpholino; wherein the alkyl between the cyclic structure and theamino may be absent, and the cyclic structures may optionally besubstituted with one or more of halo, alkoxy, hydroxy, amino,alkylamino, dialkylamino and sulfonamido.
 28. The method of claim 27whereino is 1 or 2; and M is selected from the group consisting ofalkenylamino, alkynylamino, benzylamino, and pyridinylalkylamino;wherein the cyclic structures may optionally be substituted with one ormore of halo, alkoxy, hydroxy, amino, alkylamino and dialkylamino. 29.The method of claim 28 wherein M is selected from the group consistingof alkenylamino, alkynylamino, benzylamino, and pyridinylalkylamino,wherein the cyclic structures may optionally be substituted with one ormore of halo, alkoxy, hydroxy, amino, alkylamino and dialkylamino. 30.The method of claim 29 wherein M is selected from benzylamino andbenzylamino substituted with one or more of halo, alkoxy, hydroxy,amino, alkylamino and dialkylamino.
 31. The method of claim 29 whereinRand R₁ are independently selected from the group consisting of hydrogenand hydroxy; and at least two of R₅, R₆ and R₇ are lower alkoxy and thethird is selected from hydroxy, lower alkoxy, amino and lowerdialkylamino; or each of R₅, R₆ and R₇ are hydroxy.
 32. The method ofclaim 31 whereinR and R₁ are hydrogen; R₂, R₃ and R₄ are independentlyselected from the group consisting of hydrogen, hydroxy, halogen, loweralkoxy, lower alkyl and alkyl mercapto; at least two of R₅, R₆ and R₇are lower alkoxy and the third is selected from hydroxy and loweralkoxy; R₈ is lower alkyl o is 1; and M is selected from the groupconsisting of alkenylamino, alkynylamino, benzylamino, andpyridinylalkylamino; wherein the cyclic structures may optionally besubstituted with one or more of halo, alkoxy, hydroxy, amino, alkylaminoand dialkyl amino.
 33. The method of claim 32 whereinR₅, R₆ and R₇ areeach lower alkoxy; and R₈ is methyl.
 34. The method of claim 33whereinR₂ is selected from the group consisting of hydroxy, halogen,lower alkoxy and alkyl mercapto; R₄ is hydrogen; and R₅, R₆ and R₇ areeach methoxy.
 35. The method of claim 34 whereinR₂ is selected from thegroup consisting of hydroxy, halogen and lower alkoxy; and R₃ ishydrogen.
 36. The method of claim 35 wherein R₂ is halogen.
 37. Themethod of claim 36 wherein the compound is(Z)-5-fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-(N-benzyl)-indenylacetamide.38. A method for regulating apoptosis in human cells comprising exposingsaid cells to an effective amount of a compound of the formula: ##STR5##wherein R and R₁ are independently selected from the group consisting ofhydrogen, hydroxy, lower alkyl and amino; or R and R₁ together may beoxygen;R₂, R₃ and R₄ are independently selected from the groupconsisting of hydrogen, hydroxy, halogen, lower alkoxy, lower alkyl andalkyl mercapto; at least two of R₅, R₆ and R₇ are identically selectedfrom the group consisting of hydroxy and lower alkoxy, and the third isselected from the group consisting of hydrogen, hydroxy, halogen, loweralkoxy, lower alkyl, amino and lower dialkylamino; with the proviso thatwhen at least one of R₂, R₃ or R₄ is lower alkoxy, then each of R₅, R₆and R₇ are hydroxy or lower alkoxy; R₈ is selected from the groupconsisting of hydrogen and lower alkyl; o is 0, 1 or 2; and M isselected from the group consisting of amino, alkylamino, dialkylamino,alkoxyamino, alkenylamino, alkynylamino, hydroxyalkylamino,polyhydroxyalkyl-amino, dialkylaminoalkylamino, aminoalkylamino,benzylamino, anilino, phenylalkylamino, aminoindan, andheterocycloalkylamino where the heterocycles are selected from the groupconsisting of pyridinyl, piperidinyl, piperazinyl, pyrrollidinyl andN-morpholino; wherein the alkyl between the cyclic structure and theamino may be absent, and the cyclic structures may optionally besubstituted with one or more of halo, alkoxy, hydroxy, amino,alkylamino, dialkylamino and sulfonamido.
 39. The method of claim 38whereino is 1 or 2; and M is selected from the group consisting ofalkenylamino, alkynylamino, benzylamino, and pyridinylalkylamino;wherein the cyclic structures may optionally be substituted with one ormore of halo, alkoxy, hydroxy, amino, alkylamino and dialkylamino. 40.The method of claim 39 wherein M is selected from the group consistingof alkenylamino, alkynylamino, benzylamino, and pyridinylalkylamino;wherein the cyclic structures may optionally be substituted with one ormore of halo, alkoxy, hydroxy, amino, alkylamino and dialkylamino. 41.The method of claim 40 wherein M is selected from benzylamino andbenzylamino substituted with one or more of halo, alkoxy, hydroxy,amino, alkylamino and dialkylamino.
 42. The method of claim 40 whereinRand R₁ are independently selected from the group consisting of hydrogenand hydroxy; and at least two of R₅, R₆ and R₇ are lower alkoxy and thethird is selected from hydroxy, lower alkoxy, amino and lowerdialkylamino; or each of R₅, R₆ and R₇ are hydroxy.
 43. The method ofclaim 42 whereinR and R₁ are hydrogen; R₂, R₃ and R₄ are independentlyselected from the group consisting of hydrogen, hydroxy, halogen, loweralkoxy, lower alkyl and alkyl mercapto; at least two of R₅, R₆ and R₇are lower alkoxy and the third is selected from hydroxy and loweralkoxy; R₈ is lower alkyl; o is 1; and M is selected from the groupconsisting of alkenylamino, alkynylamino, benzylamino, andpyridinylalkylamino; wherein the cyclic structures may optionally besubstituted with one or more of halo, alkoxy, hydroxy, amino, alkylamino and dialkylamino.
 44. The method of claim 43 whereinR₅, R₆ and R₇are each lower alkoxy; and R₈ is methyl.
 45. The method of claim 44whereinR₂ is selected from the group consisting of hydroxy, halogen,lower alkoxy and alkyl mercapto; R₄ is hydrogen; and R₅, R₆ and R₇ areeach methoxy.
 46. The method, of claim 45 whereinR₂ is selected from thegroup consisting of hydroxy, halogen and lower alkoxy, and R₃ ishydrogen.
 47. The method of claim 46 wherein R₂ is fluoro.